高效表达重组HIV-1膜蛋白gp41及其在尿液检测中的应用
摘要
人获得性免疫缺陷综合症(Acquired Immunodeficiency Syndrome,AIDS),简称艾滋病,是由人免疫缺陷病毒(Human Immunodeficiency Virus,HIV)引起的。自1983年中国出现首例HIV感染者至2007年10月底,中国累计报告艾滋病病毒感染者223501例,其中艾滋病病人62838例,死亡报告22205人。尽管经过20多年的研究,在HIV基础研究和预防治疗方面取得了较大进展,但是现在仍然没有彻底治愈AIDS的手段和令人满意的疫苗。目前控制HIV流行的主要方法是仍然是行为干预。因此,及早发现HIV感染病例对控制HIV流行起着举足轻重的作用。
HIV感染人体后,患者体内可产生针对HIV结构蛋白的特异性抗体,包括核心蛋白(P24、P17);膜蛋白(HIV-1:Gp160、Gp120、Gp41;HIV-2:Gp140、Gp105、Gp36)以及病毒酶蛋白(P66、P31)的抗体。其中外膜蛋白的抗体在病人一经产生就一直维持较高水平,因此,检测HIV—1膜蛋白的抗体,特别是gp41的抗体是判断是否感染HIV的标准之一。
常规HIV检测通常是以血液为检测对象,近年来以尿液、口腔渗出液为样本的HIV检测试剂逐渐受到关注。与血液检测试剂相比,这些检测试剂具有无创、无传染性等优点,在快速检测、研究同性恋等特定人群的HIV感染情况等领域得到了一定程度的应用。与血液检测试剂相比,因为尿液中的HIV特异性抗体浓度低,尿液HIV检测试剂对使用的抗原的质量要求更高。
本文以研制高质量HIV-1 gp41诊断用抗原,制备HIV—1尿液检测试剂为目的,包括如下几个方面的内容:
1、重组gp41的基因优化及原核表达质粒的构建:在本室保存的HIV-1 gp41表达质粒pGEX-5X-1-rgp41的基础上,用长引物多聚酶链式反应(polymerasechain reaction,PCR)法对gp41基因上游80bp进行大肠杆菌偏爱密码子优化,经过2轮PCR反应完成了基因优化并在基因上下游引入了酶切位点;然后将优化后的基因分别插入到原核表达载体pTXB、pET32a、pET22b中,构建原核表达质粒。
2、重组gp41的表达和纯化:分别将上述构建的原核表达质粒进行表达筛选;最终选用pET22b-mgp41进行大量表达和纯化,使用破菌、洗涤包涵体、金属螯合层析和分子筛层析等步骤纯化重组Gp41,最终得到的重组Gp41纯度大于95%。
3、重组Gp41大量发酵的工艺摸索:使用小型发酵罐对重组表达菌的大量发酵工艺进行了摸索,通过优化细菌生长条件,2.5L的培养物经过8小时的培养和诱导表达最终可以得到120g湿菌,目标蛋白的表达量达到25%以上。
4、尿液HIV—1抗体检测方法的初步建立和样品考核:使用本研究中制备的重组Gp41抗原,制备尿液HIV-1抗体检测试剂盒,并用临床样本对试剂盒进行了样品考核。考核结果显示,本研究建立的HIV-1抗体尿液检测方法的灵敏度和特异性分别达到了100%和98.52%,可以满足HIV-1初筛实验的要求。
Human acquired immune deficiency syndrome (AIDS), is caused by a retrovirus-human immunodeficiency virus (HIV). Under the conditions there is no effective vaccine and curable treatment for AIDS, it is an important strategy to detect the HIV antibodies among the high risk population and blood donors to prevent and control the spread of AIDS by interrupting the ways for transmission.
Testing for HIV is an essential component in the diagnosis and treatment of persons infected with the virus, in screening of blood for transfusion, in surveillance and in HIV/AIDS related research. Thus accurate and cost-effective testing is of great importance in combating the spread of HIV. It is imperative that tests for the diagnosis of HIV infection be as accurate as possible, given the serious ethical, legal and social issues that accompany HIV infection. The use of body fluids other than blood as specimens for detecting antibodies to HIV has been reported to have potential as an alternative medium for HIV testing. The use of urine samples and also samples derived from the oral cavity (saliva) may be attractive due to the ease of sample collection, possible cost savings, better safety (against needlestick injuries) and higher compliance rates. Assays for these types of specimens can be useful alternative when it is difficult or impossible to test for HIV in blood samples. It may be that blood cannot be drawn for religious reasons or difficulties may be experienced in collecting blood samples in hard to reach places where it is, nevertheless, important to have epidemiological surveillance.
To establish a specific and sensitive Enzyme-linked immunosorbent Assay(ELISA) kit for detection of HIV-1 antibody in urine using Escherichia coli expression products as coating antigen is the main objective of this study. The truncated HIV-1 gp41 gene fragment containing major antigenic epitopes was inserted into the plasmid pET22b to obtain expression plasmid pET22b-mgp41. The recombinant antigen was expressed in BL21(DE3) strains of Escherichia coli and was purified by immobilized metal chelation and gel filtration chromatography. The purity of purified antigen is up to 95%. Using this antigen as coating antigen, a HIV-1 urine antibody ELISA kit was developed. In order to examine the clinical utility of the kit, 5437 urine samples were assayed, which consisted of 641 urine samples from HIV infected patients and 4796 samples from normal subjects. Anti-HIV antibodies were detected in all the urine samples from HIV infected patients, and the diagnostic sensitivity for HIV-1 infection was 100%. In healthy control group, 71 cases showed false positive, the specificity was 98.52%. The HIV-1 urine antibody kit can be used in screening and diagnosing for HIV-1 infection.
HIV感染人体后,患者体内可产生针对HIV结构蛋白的特异性抗体,包括核心蛋白(P24、P17);膜蛋白(HIV-1:Gp160、Gp120、Gp41;HIV-2:Gp140、Gp105、Gp36)以及病毒酶蛋白(P66、P31)的抗体。其中外膜蛋白的抗体在病人一经产生就一直维持较高水平,因此,检测HIV—1膜蛋白的抗体,特别是gp41的抗体是判断是否感染HIV的标准之一。
常规HIV检测通常是以血液为检测对象,近年来以尿液、口腔渗出液为样本的HIV检测试剂逐渐受到关注。与血液检测试剂相比,这些检测试剂具有无创、无传染性等优点,在快速检测、研究同性恋等特定人群的HIV感染情况等领域得到了一定程度的应用。与血液检测试剂相比,因为尿液中的HIV特异性抗体浓度低,尿液HIV检测试剂对使用的抗原的质量要求更高。
本文以研制高质量HIV-1 gp41诊断用抗原,制备HIV—1尿液检测试剂为目的,包括如下几个方面的内容:
1、重组gp41的基因优化及原核表达质粒的构建:在本室保存的HIV-1 gp41表达质粒pGEX-5X-1-rgp41的基础上,用长引物多聚酶链式反应(polymerasechain reaction,PCR)法对gp41基因上游80bp进行大肠杆菌偏爱密码子优化,经过2轮PCR反应完成了基因优化并在基因上下游引入了酶切位点;然后将优化后的基因分别插入到原核表达载体pTXB、pET32a、pET22b中,构建原核表达质粒。
2、重组gp41的表达和纯化:分别将上述构建的原核表达质粒进行表达筛选;最终选用pET22b-mgp41进行大量表达和纯化,使用破菌、洗涤包涵体、金属螯合层析和分子筛层析等步骤纯化重组Gp41,最终得到的重组Gp41纯度大于95%。
3、重组Gp41大量发酵的工艺摸索:使用小型发酵罐对重组表达菌的大量发酵工艺进行了摸索,通过优化细菌生长条件,2.5L的培养物经过8小时的培养和诱导表达最终可以得到120g湿菌,目标蛋白的表达量达到25%以上。
4、尿液HIV—1抗体检测方法的初步建立和样品考核:使用本研究中制备的重组Gp41抗原,制备尿液HIV-1抗体检测试剂盒,并用临床样本对试剂盒进行了样品考核。考核结果显示,本研究建立的HIV-1抗体尿液检测方法的灵敏度和特异性分别达到了100%和98.52%,可以满足HIV-1初筛实验的要求。
Human acquired immune deficiency syndrome (AIDS), is caused by a retrovirus-human immunodeficiency virus (HIV). Under the conditions there is no effective vaccine and curable treatment for AIDS, it is an important strategy to detect the HIV antibodies among the high risk population and blood donors to prevent and control the spread of AIDS by interrupting the ways for transmission.
Testing for HIV is an essential component in the diagnosis and treatment of persons infected with the virus, in screening of blood for transfusion, in surveillance and in HIV/AIDS related research. Thus accurate and cost-effective testing is of great importance in combating the spread of HIV. It is imperative that tests for the diagnosis of HIV infection be as accurate as possible, given the serious ethical, legal and social issues that accompany HIV infection. The use of body fluids other than blood as specimens for detecting antibodies to HIV has been reported to have potential as an alternative medium for HIV testing. The use of urine samples and also samples derived from the oral cavity (saliva) may be attractive due to the ease of sample collection, possible cost savings, better safety (against needlestick injuries) and higher compliance rates. Assays for these types of specimens can be useful alternative when it is difficult or impossible to test for HIV in blood samples. It may be that blood cannot be drawn for religious reasons or difficulties may be experienced in collecting blood samples in hard to reach places where it is, nevertheless, important to have epidemiological surveillance.
To establish a specific and sensitive Enzyme-linked immunosorbent Assay(ELISA) kit for detection of HIV-1 antibody in urine using Escherichia coli expression products as coating antigen is the main objective of this study. The truncated HIV-1 gp41 gene fragment containing major antigenic epitopes was inserted into the plasmid pET22b to obtain expression plasmid pET22b-mgp41. The recombinant antigen was expressed in BL21(DE3) strains of Escherichia coli and was purified by immobilized metal chelation and gel filtration chromatography. The purity of purified antigen is up to 95%. Using this antigen as coating antigen, a HIV-1 urine antibody ELISA kit was developed. In order to examine the clinical utility of the kit, 5437 urine samples were assayed, which consisted of 641 urine samples from HIV infected patients and 4796 samples from normal subjects. Anti-HIV antibodies were detected in all the urine samples from HIV infected patients, and the diagnostic sensitivity for HIV-1 infection was 100%. In healthy control group, 71 cases showed false positive, the specificity was 98.52%. The HIV-1 urine antibody kit can be used in screening and diagnosing for HIV-1 infection.
引文
1 Zeng Y, Fan J, Zhang Q, et al. Detection of antibody to LAV/HTLV-III in sera from hemophiliacs in China. AIDS Res, 1986,2 Suppl l:S147-9.
2 中国艾滋病防治联合评估报告(2007年).
3 Hernandez Marin M, Marquez Bocalandro Y, VallejoRV, et al. Use of a chimeric synthetic peptide from the core pl9 protein and the envelope gp46 glycoprotein in the immunodiagnosis of HTLV-II virus infection. Prep Biochem Biotechnol, 2003,33:29-38.
4 Tenner-Racz K, Racz P, Bofill M, et al. HTLV-III/LAV viral antigens in lymph nodes of homosexual men with persistent generalized lymphadenopathy and AIDS. Am J Pathol,1986,123:9-15.
5 Truett M A, Chein D Y, Calarco T L, et al. HIV detection by genetic engineering method, 1989.41-47.
6 Cao Y, Friedman-Kien AE, Chuba JV, et al. IgG antibodies to HIV-1 in urine of HIV-1 seropositive individuals. Lancet JT -Lancet, 1988,1:831-2.
7 Abrao Ferreira PR, Gabriel R, Furlan TM, et al. Anti-HIV-1/2 Antibody Detection by Dot-ELISA in Oral Fluid of HIV Positive/AIDS Patients and Voluntary Blood Donors. Braz J Infect Dis,1999,3:134-138.
8 Matee MI, Lyamuya EF, Simon E, et al. Detection of anti-HIV-1 IgG antibodies in whole saliva by GACELISA and Western blot assays. East Afr Med J JT - East African medical journal,1996,73:292-4.
9 Desai S, Bates H, Michalski FJ. Detection of antibody to HIV-1 in urine. Lancet JT -Lancet,1991,337:183-4.
10 Reagan KJ, Lile CC, Book GW, et al. Use of urine for HIV-1 antibody screening. Lancet JT-Lancet, 1990,335:358-9.
11 Lee SS, Lee KC, Wan WY, et al. A urine-based approach to scale up HIV testing in drug users.AIDS, 2006,20:1349-51.
12 Hilton C, Sabundayo BP, Langan SJ, et al. Screening for HIV infection in high-risk communities by urine antibody testing. J Acquir Immune Defic Syndr JT - Journal of acquired immune deficiency syndromes (1999), 2002,31:416-21.
13 Peralta L, Constantine N, Griffin Deeds B, et al. Evaluation of youth preferences for rapid and innovative human immunodeficiency virus antibody tests. Arch Pediatr Adolesc Med JT -Archives of pediatrics & adolescent medicine, 2001,155:838-43.
14 Muralidharan S, Srinivasa H, Ravindran GD, et al. Urinary IgG antibody capture particles adherence test for detecting HIV antibodies: a preliminary report. J Assoc Physicians India JT - The Journal of the Association of Physicians of India, 1998,46:699-700.
15 Stevens RW, Baltch AL, Smith RP, et al. Antibody to human endogenous retrovirus peptide in urine of human immunodeficiency virus type 1-positive patients. Clin Diagn Lab Immunol JT- Clinical and diagnostic laboratory immunology, 1999,6:783-6.
16 Urnovitz HB, Sturge JC, Gottfried TD, et al. Urine antibody tests: new insights into the dynamics of HIV-1 infection. Clin Chem JT - Clinical chemistry, 1999,45:1602-13.
17 Martinez PM, Torres AR, Ortiz de Lejarazu R, et al. Human immunodeficiency virus antibody testing by enzyme-linked fluorescent and western blot assays using serum,gingival-crevicular transudate, and urine samples. J Clin Microbiol JT - Journal of clinical microbiology, 1999,37:1100-6.
18 Hashida S, Hashinaka K, Ishikawa S, et al. More reliable diagnosis of infection with human immunodeficiency virus type 1 (HIV-1) by detection of antibody IgGs to pol and gag proteins of HIV-1 and p24 antigen of HIV-1 in urine, saliva, and/or serum with highly sensitive and specific enzyme immunoassay (immune complex transfer enzyme immunoassay): a review. J Clin Lab Anal JT - Journal of clinical laboratory analysis, 1997,11:267-86.
19 Connell JA, Parry JV, Mortimer PP, et al. Preliminary report: accurate assays for anti-HIV in urine. Lancet JT - Lancet, 1990,335:1366-9.
20 Kim S, Lee SB. Rare codon clusters at 5' -end influence heterologous expression of archaeal gene in Escherichia coli. Protein Expr Purif JT - Protein expression and purification,2006,50:49-57.
21 Qian B, Shen H, Xiong J, et al. Expression and purification of the synthetic preS1 gene of Hepatitis B Virus with preferred Escherichia coli codon preference. Protein Expr Purif JT- Protein expression and purification, 2006,48:74-80.
22 Popovic M, Coglievina M, Guarnaccia C, et al. Gene synthesis, expression, purification, and characterization of human Jagged-1 intracellular region. Protein Expr Purif JT - Protein expression and purification, 2006,47:398-404.
23 Delroisse JM, Dannau M, Gilsoul JJ, et al. Expression of a synthetic gene encoding a Tribolium castaneum carboxylesterase in Pichia pastoris. Protein Expr Purif JT - Protein expression and purification, 2005,42:286-94.
24 Scorer CA, Buckholz RG, Clare JJ, et al. The intracellular production and secretion of HIV-1 envelope protein in the methylotrophic yeast Pichia pastoris. Gene JT - Gene,1993, 136:111-9.
25 袁玉华,毕昌昊,李菊,王学谦,耿运琪,陈启民.HIV gp41基因分段低温诱导表达.中国病毒学,2004, 19:179-181.
26 Ebenbichler CF, Roder C, Vornhagen R, et al. Cell surface proteins binding to recombinant soluble HIV-1 and HIV-2 transmembrane proteins. AIDS, 1993,7:489-95.
27 Hofbauer JM, Schulz TF, Hengster P, et al. Comparison of Western blot (immunoblot) based on recombinant-derived p41 with conventional tests for serodiagnosis of human immunodeficiency virus infections. J Clin Microbiol, 1988,26:116-20.
28 Sohn MJ, Lee ME, Park HS, et al. Overexpression and purification of human immunodeficiency virus type 1 env derived epitopes in Escherichia coli. J Biotechnol, 1996,45:211-6.
29 Mangavel C, Maget-Dana R, Tauc P, et al. Structural investigations of basic amphipathic model peptides in the presence of lipid vesicles studied by circular dichroism, fluorescence,monolayer and modeling. Biochim Biophys Acta, 1998,1371:265-83.
30 Boycheva S, Chkodrov G, Ivanov I. Codon pairs in the genome of Escherichia coli.Bioinformatics JT - Bioinformatics (Oxford, England), 2003,19:987-98.
31 Griswold KE, Mahmood NA, Iverson BL, et al. Effects of codon usage versus putative 5'-mRNA structure on the expression of Fusarium solani cutinase in the Escherichia coli cytoplasm.Protein Expr Purif JT - Protein expression and purification, 2003,27:134-42.
32 Acosta-Rivero N, Sanchez JC, Morales J. Improvement of human interferon HUIFNalpha2 and HCV core protein expression levels in Escherichia coli but not of HUIFNalpha8 by using the tRNA (AGA/AGG). Biochem Biophys Res Commun JT - Biochemical and biophysical research communications, 2002,296:1303-9.
33 Areas AP, Oliveira ML, Ramos CR, et al. Synthesis of cholera toxin B subunit gene: cloning and expression of a functional 6XHis-tagged protein in Escherichia coli. Protein Expr Purif JT - Protein expression and purification, 2002,25:481-7.
34 Li Y, Chen CX, von Specht BU, et al. Cloning and hemolysin-mediated secretory expression of a codon-optimized synthetic human interleukin-6 gene in Escherichia coli. Protein Expr Purif JT - Protein expression and purification, 2002,25:437-47.
35 Gao F, Li Y, Decker JM, et al. Codon usage optimization of HIV type 1 subtype C gag, pol,env, and nef genes: in vitro expression and immune responses in DNA-vaccinated mice. AIDS Res Hum Retroviruses JT - AIDS research and human retroviruses, 2003, 19:817-23.
36 McHardy AC, Puhler A, Kalinowski J, et al. Comparing expression level-dependent features in codon usage with protein abundance: an analysis of 'predictive proteomics'. Proteomics JT -Proteomics, 2004,4:46-58.
37 Lakey DL, Voladri RK, Edwards KM, et al. Enhanced production of recombinant Mycobacterium tuberculosis antigens in Escherichia coli by replacement of low-usage codons. Infect Immun JT - Infection and immunity, 2000,68:233-8.
38 Holler TP, Foltin SK, Ye QZ, et al. HIV1 integrase expressed in Escherichia coli from a synthetic gene. Gene JT - Gene, 1993, 136:323-8.
39 Bauer R, Himly M, Dedic A, et al. Optimization of codon usage is required for effective genetic immunization against Art v 1, the major allergen of mugwort pollen. Allergy JT - Allergy,2003,58:1003-10.
40 Karlin S, Mrazek J. Predicted highly expressed genes of diverse prokaryotic genomes. J Bacteriol JT - Journal of bacteriology, 2000,182:5238-50.
41 Zhang W, Xiao W, Wei H, et al. mRNA secondary structure at start AUG codon is a key limiting factor for human protein expression in Escherichia coli. Biochem Biophys Res Commun JT -Biochemical and biophysical research communications, 2006,349:69-78.
42 Looman AC,Bodlaender J,Comstock LJ,et al.Influence of the codon following the AUG initiation codon on the expression of a modified lacZ gene in Escherichia coli.EMBO J JT -The EMBO journal,1987,6:2489-92.
43 萨姆布鲁克 J,弗里奇E F,曼尼阿蒂斯T.分子克隆实验指南.第三版版.北京:科学出版社,2002.
44 Zhao Z,Lu W,Dun B,et al.Purification of green fluorescent protein using a two-intein system.Appl Microbiol Biotechnol,2008,77:1175-80.
45 Gillies AR,Mahmoud RB,Wood DW.PHB-Intein-Mediated Protein Purification Strategy.Methods Mol Biol,2009,498:173-83.
46 Hong J,Wang Y,Ye X,et al.Simple protein purification through affinity adsorption on regenerated amorphous cellulose followed by intein self-cleavage.J Chromatogr A,2008,1194:150-4.
47 Esipov RS,Stepanenko VN,Chupova LA,et al.Production of recombinant human epidermal growth factor using Ssp dnaB mini-intein system.Protein Expr Purif,2008,61:1-6.
48 Cantor EJ,Chong S.Intein-mediated rapid purification of Cre recombinase.Protein Expr Purif,2001,22:135-40.
49 Tiensiwakul P.Urinary HIM-1 antibody patterns by western blot assay.Clin Lab Sci JT-Clinical laboratory science:journal of the American Society for Medical Technology,1998,11:336-8.
50 Urnovitz HB,Murphy WH,Gottfried TD,et al.Urine-based diagnostic technologies.Trends Biotechnol JT-Trends in biotechnology,1996,14:361-4.
51 Martinez P,Lejarazu RO,Eiros JM,et al.Urine samples as a possible alternative to serum for human immunodeficiency virus antibody screening.Eur J Clin Microbiol Infect Dis JT-European journal of clinical microbiology & infectious diseases:official publication of the European Society of Clinical Microbiology,1996,15:810-3.
52 赵立庆,雷素萍,李连学,赵金仙,代昆玉,陈良,鲁建波,李孝安,普学云.ELISA法检测尿液中HIV_1抗体的研究分析.中国艾滋病性病,2003,9:276-277,269.
53 伦文辉 李莉 黄钦 李敏 曹韵贞.ELISA榆测HIV感染者尿液标本中抗HIV_1抗体结果分析.中国性病艾滋病防治,2001,7:32-34.
54 黄帧祥,洪涛,刘崇柏.医学病毒学基础及实验技术:科学出版社,1990.
55 尿液中抗HIV-1抗体的研究.中国性病艾滋病防治,1995,5:33-35.
56 Martinez PM,Torres AR,Ortiz de Lejarazu R,et al.Human immunodeficiency virus antibody testing by enzyme-linked fluorescent and western blot assays using serum,gingival-crevicular transudate,and urine samples.J Clin Microbiol,1999,37:1100-6.
57 Tiensiwakul P.Urinary HIV-1 antibody patterns by western blot assay.Clin Lab Sci,1998,11:336-8.
58 Allan JS,Coligan JE,Barin F,et al.Major glycoprotein antigens that induce antibodies in AIDS patients are encoded by HTLV-III.Science,1985,228:1091-4.
59 Horal P,Svennerholm B,Jeansson S,et al.Continuous epitopes of the human immunodeficiency virus type 1(HIV-1) transmembrane glycoprotein and reactivity of human sera to synthetic peptides representing various HIV-1 isolates.J Virol,1991,65:2718-23.
60 Hayashi T,Watanabe S,Kondo M,et al.[Evaluation of a new screening assay kit for the combined detection of HIV p24 antigen and antibody--comparison of the performance of the new kit and HIV antibody assay kits].Kansenshogaku Zasshi,1999,73:681-8.
61 Hayashi T,Saito T,Kondo M,et al.[Investigation of a new HIV-1 p24 antigen detection kit based on the enzyme-linked fluorescent immunoassay].Kansenshogaku Zasshi,2000,74:709-15.
62 Martinez-Martinez P,Martin del Barrio E,De Benito J,et al.New lineal immunoenzymatic assay for simultaneous detection of p24 antigen and HIV antibodies.Eur J Clin Microbiol Infect Dis,1999,18:591-4.
63 Kumar P,Mui HX,Kappes JC,et al.Molecular characterization of an attenuated human immunodeficiency virus type 2 isolate.J Virol,1990,64:890-901.
64 黑发欣,张启云,孙伟东,等.HIV抗体筛查实验阳性确认实验可疑和阴性标本的对比分析.中国性病艾滋病,2006,12:4-6.
65 Cai F,Chen H,Hicks CB,et al.Detection of minor drug-resistant populations by parallel allele-specific sequencing.Nat Methods,2007,4:123-5.
2 中国艾滋病防治联合评估报告(2007年).
3 Hernandez Marin M, Marquez Bocalandro Y, VallejoRV, et al. Use of a chimeric synthetic peptide from the core pl9 protein and the envelope gp46 glycoprotein in the immunodiagnosis of HTLV-II virus infection. Prep Biochem Biotechnol, 2003,33:29-38.
4 Tenner-Racz K, Racz P, Bofill M, et al. HTLV-III/LAV viral antigens in lymph nodes of homosexual men with persistent generalized lymphadenopathy and AIDS. Am J Pathol,1986,123:9-15.
5 Truett M A, Chein D Y, Calarco T L, et al. HIV detection by genetic engineering method, 1989.41-47.
6 Cao Y, Friedman-Kien AE, Chuba JV, et al. IgG antibodies to HIV-1 in urine of HIV-1 seropositive individuals. Lancet JT -Lancet, 1988,1:831-2.
7 Abrao Ferreira PR, Gabriel R, Furlan TM, et al. Anti-HIV-1/2 Antibody Detection by Dot-ELISA in Oral Fluid of HIV Positive/AIDS Patients and Voluntary Blood Donors. Braz J Infect Dis,1999,3:134-138.
8 Matee MI, Lyamuya EF, Simon E, et al. Detection of anti-HIV-1 IgG antibodies in whole saliva by GACELISA and Western blot assays. East Afr Med J JT - East African medical journal,1996,73:292-4.
9 Desai S, Bates H, Michalski FJ. Detection of antibody to HIV-1 in urine. Lancet JT -Lancet,1991,337:183-4.
10 Reagan KJ, Lile CC, Book GW, et al. Use of urine for HIV-1 antibody screening. Lancet JT-Lancet, 1990,335:358-9.
11 Lee SS, Lee KC, Wan WY, et al. A urine-based approach to scale up HIV testing in drug users.AIDS, 2006,20:1349-51.
12 Hilton C, Sabundayo BP, Langan SJ, et al. Screening for HIV infection in high-risk communities by urine antibody testing. J Acquir Immune Defic Syndr JT - Journal of acquired immune deficiency syndromes (1999), 2002,31:416-21.
13 Peralta L, Constantine N, Griffin Deeds B, et al. Evaluation of youth preferences for rapid and innovative human immunodeficiency virus antibody tests. Arch Pediatr Adolesc Med JT -Archives of pediatrics & adolescent medicine, 2001,155:838-43.
14 Muralidharan S, Srinivasa H, Ravindran GD, et al. Urinary IgG antibody capture particles adherence test for detecting HIV antibodies: a preliminary report. J Assoc Physicians India JT - The Journal of the Association of Physicians of India, 1998,46:699-700.
15 Stevens RW, Baltch AL, Smith RP, et al. Antibody to human endogenous retrovirus peptide in urine of human immunodeficiency virus type 1-positive patients. Clin Diagn Lab Immunol JT- Clinical and diagnostic laboratory immunology, 1999,6:783-6.
16 Urnovitz HB, Sturge JC, Gottfried TD, et al. Urine antibody tests: new insights into the dynamics of HIV-1 infection. Clin Chem JT - Clinical chemistry, 1999,45:1602-13.
17 Martinez PM, Torres AR, Ortiz de Lejarazu R, et al. Human immunodeficiency virus antibody testing by enzyme-linked fluorescent and western blot assays using serum,gingival-crevicular transudate, and urine samples. J Clin Microbiol JT - Journal of clinical microbiology, 1999,37:1100-6.
18 Hashida S, Hashinaka K, Ishikawa S, et al. More reliable diagnosis of infection with human immunodeficiency virus type 1 (HIV-1) by detection of antibody IgGs to pol and gag proteins of HIV-1 and p24 antigen of HIV-1 in urine, saliva, and/or serum with highly sensitive and specific enzyme immunoassay (immune complex transfer enzyme immunoassay): a review. J Clin Lab Anal JT - Journal of clinical laboratory analysis, 1997,11:267-86.
19 Connell JA, Parry JV, Mortimer PP, et al. Preliminary report: accurate assays for anti-HIV in urine. Lancet JT - Lancet, 1990,335:1366-9.
20 Kim S, Lee SB. Rare codon clusters at 5' -end influence heterologous expression of archaeal gene in Escherichia coli. Protein Expr Purif JT - Protein expression and purification,2006,50:49-57.
21 Qian B, Shen H, Xiong J, et al. Expression and purification of the synthetic preS1 gene of Hepatitis B Virus with preferred Escherichia coli codon preference. Protein Expr Purif JT- Protein expression and purification, 2006,48:74-80.
22 Popovic M, Coglievina M, Guarnaccia C, et al. Gene synthesis, expression, purification, and characterization of human Jagged-1 intracellular region. Protein Expr Purif JT - Protein expression and purification, 2006,47:398-404.
23 Delroisse JM, Dannau M, Gilsoul JJ, et al. Expression of a synthetic gene encoding a Tribolium castaneum carboxylesterase in Pichia pastoris. Protein Expr Purif JT - Protein expression and purification, 2005,42:286-94.
24 Scorer CA, Buckholz RG, Clare JJ, et al. The intracellular production and secretion of HIV-1 envelope protein in the methylotrophic yeast Pichia pastoris. Gene JT - Gene,1993, 136:111-9.
25 袁玉华,毕昌昊,李菊,王学谦,耿运琪,陈启民.HIV gp41基因分段低温诱导表达.中国病毒学,2004, 19:179-181.
26 Ebenbichler CF, Roder C, Vornhagen R, et al. Cell surface proteins binding to recombinant soluble HIV-1 and HIV-2 transmembrane proteins. AIDS, 1993,7:489-95.
27 Hofbauer JM, Schulz TF, Hengster P, et al. Comparison of Western blot (immunoblot) based on recombinant-derived p41 with conventional tests for serodiagnosis of human immunodeficiency virus infections. J Clin Microbiol, 1988,26:116-20.
28 Sohn MJ, Lee ME, Park HS, et al. Overexpression and purification of human immunodeficiency virus type 1 env derived epitopes in Escherichia coli. J Biotechnol, 1996,45:211-6.
29 Mangavel C, Maget-Dana R, Tauc P, et al. Structural investigations of basic amphipathic model peptides in the presence of lipid vesicles studied by circular dichroism, fluorescence,monolayer and modeling. Biochim Biophys Acta, 1998,1371:265-83.
30 Boycheva S, Chkodrov G, Ivanov I. Codon pairs in the genome of Escherichia coli.Bioinformatics JT - Bioinformatics (Oxford, England), 2003,19:987-98.
31 Griswold KE, Mahmood NA, Iverson BL, et al. Effects of codon usage versus putative 5'-mRNA structure on the expression of Fusarium solani cutinase in the Escherichia coli cytoplasm.Protein Expr Purif JT - Protein expression and purification, 2003,27:134-42.
32 Acosta-Rivero N, Sanchez JC, Morales J. Improvement of human interferon HUIFNalpha2 and HCV core protein expression levels in Escherichia coli but not of HUIFNalpha8 by using the tRNA (AGA/AGG). Biochem Biophys Res Commun JT - Biochemical and biophysical research communications, 2002,296:1303-9.
33 Areas AP, Oliveira ML, Ramos CR, et al. Synthesis of cholera toxin B subunit gene: cloning and expression of a functional 6XHis-tagged protein in Escherichia coli. Protein Expr Purif JT - Protein expression and purification, 2002,25:481-7.
34 Li Y, Chen CX, von Specht BU, et al. Cloning and hemolysin-mediated secretory expression of a codon-optimized synthetic human interleukin-6 gene in Escherichia coli. Protein Expr Purif JT - Protein expression and purification, 2002,25:437-47.
35 Gao F, Li Y, Decker JM, et al. Codon usage optimization of HIV type 1 subtype C gag, pol,env, and nef genes: in vitro expression and immune responses in DNA-vaccinated mice. AIDS Res Hum Retroviruses JT - AIDS research and human retroviruses, 2003, 19:817-23.
36 McHardy AC, Puhler A, Kalinowski J, et al. Comparing expression level-dependent features in codon usage with protein abundance: an analysis of 'predictive proteomics'. Proteomics JT -Proteomics, 2004,4:46-58.
37 Lakey DL, Voladri RK, Edwards KM, et al. Enhanced production of recombinant Mycobacterium tuberculosis antigens in Escherichia coli by replacement of low-usage codons. Infect Immun JT - Infection and immunity, 2000,68:233-8.
38 Holler TP, Foltin SK, Ye QZ, et al. HIV1 integrase expressed in Escherichia coli from a synthetic gene. Gene JT - Gene, 1993, 136:323-8.
39 Bauer R, Himly M, Dedic A, et al. Optimization of codon usage is required for effective genetic immunization against Art v 1, the major allergen of mugwort pollen. Allergy JT - Allergy,2003,58:1003-10.
40 Karlin S, Mrazek J. Predicted highly expressed genes of diverse prokaryotic genomes. J Bacteriol JT - Journal of bacteriology, 2000,182:5238-50.
41 Zhang W, Xiao W, Wei H, et al. mRNA secondary structure at start AUG codon is a key limiting factor for human protein expression in Escherichia coli. Biochem Biophys Res Commun JT -Biochemical and biophysical research communications, 2006,349:69-78.
42 Looman AC,Bodlaender J,Comstock LJ,et al.Influence of the codon following the AUG initiation codon on the expression of a modified lacZ gene in Escherichia coli.EMBO J JT -The EMBO journal,1987,6:2489-92.
43 萨姆布鲁克 J,弗里奇E F,曼尼阿蒂斯T.分子克隆实验指南.第三版版.北京:科学出版社,2002.
44 Zhao Z,Lu W,Dun B,et al.Purification of green fluorescent protein using a two-intein system.Appl Microbiol Biotechnol,2008,77:1175-80.
45 Gillies AR,Mahmoud RB,Wood DW.PHB-Intein-Mediated Protein Purification Strategy.Methods Mol Biol,2009,498:173-83.
46 Hong J,Wang Y,Ye X,et al.Simple protein purification through affinity adsorption on regenerated amorphous cellulose followed by intein self-cleavage.J Chromatogr A,2008,1194:150-4.
47 Esipov RS,Stepanenko VN,Chupova LA,et al.Production of recombinant human epidermal growth factor using Ssp dnaB mini-intein system.Protein Expr Purif,2008,61:1-6.
48 Cantor EJ,Chong S.Intein-mediated rapid purification of Cre recombinase.Protein Expr Purif,2001,22:135-40.
49 Tiensiwakul P.Urinary HIM-1 antibody patterns by western blot assay.Clin Lab Sci JT-Clinical laboratory science:journal of the American Society for Medical Technology,1998,11:336-8.
50 Urnovitz HB,Murphy WH,Gottfried TD,et al.Urine-based diagnostic technologies.Trends Biotechnol JT-Trends in biotechnology,1996,14:361-4.
51 Martinez P,Lejarazu RO,Eiros JM,et al.Urine samples as a possible alternative to serum for human immunodeficiency virus antibody screening.Eur J Clin Microbiol Infect Dis JT-European journal of clinical microbiology & infectious diseases:official publication of the European Society of Clinical Microbiology,1996,15:810-3.
52 赵立庆,雷素萍,李连学,赵金仙,代昆玉,陈良,鲁建波,李孝安,普学云.ELISA法检测尿液中HIV_1抗体的研究分析.中国艾滋病性病,2003,9:276-277,269.
53 伦文辉 李莉 黄钦 李敏 曹韵贞.ELISA榆测HIV感染者尿液标本中抗HIV_1抗体结果分析.中国性病艾滋病防治,2001,7:32-34.
54 黄帧祥,洪涛,刘崇柏.医学病毒学基础及实验技术:科学出版社,1990.
55 尿液中抗HIV-1抗体的研究.中国性病艾滋病防治,1995,5:33-35.
56 Martinez PM,Torres AR,Ortiz de Lejarazu R,et al.Human immunodeficiency virus antibody testing by enzyme-linked fluorescent and western blot assays using serum,gingival-crevicular transudate,and urine samples.J Clin Microbiol,1999,37:1100-6.
57 Tiensiwakul P.Urinary HIV-1 antibody patterns by western blot assay.Clin Lab Sci,1998,11:336-8.
58 Allan JS,Coligan JE,Barin F,et al.Major glycoprotein antigens that induce antibodies in AIDS patients are encoded by HTLV-III.Science,1985,228:1091-4.
59 Horal P,Svennerholm B,Jeansson S,et al.Continuous epitopes of the human immunodeficiency virus type 1(HIV-1) transmembrane glycoprotein and reactivity of human sera to synthetic peptides representing various HIV-1 isolates.J Virol,1991,65:2718-23.
60 Hayashi T,Watanabe S,Kondo M,et al.[Evaluation of a new screening assay kit for the combined detection of HIV p24 antigen and antibody--comparison of the performance of the new kit and HIV antibody assay kits].Kansenshogaku Zasshi,1999,73:681-8.
61 Hayashi T,Saito T,Kondo M,et al.[Investigation of a new HIV-1 p24 antigen detection kit based on the enzyme-linked fluorescent immunoassay].Kansenshogaku Zasshi,2000,74:709-15.
62 Martinez-Martinez P,Martin del Barrio E,De Benito J,et al.New lineal immunoenzymatic assay for simultaneous detection of p24 antigen and HIV antibodies.Eur J Clin Microbiol Infect Dis,1999,18:591-4.
63 Kumar P,Mui HX,Kappes JC,et al.Molecular characterization of an attenuated human immunodeficiency virus type 2 isolate.J Virol,1990,64:890-901.
64 黑发欣,张启云,孙伟东,等.HIV抗体筛查实验阳性确认实验可疑和阴性标本的对比分析.中国性病艾滋病,2006,12:4-6.
65 Cai F,Chen H,Hicks CB,et al.Detection of minor drug-resistant populations by parallel allele-specific sequencing.Nat Methods,2007,4:123-5.