禽流感病毒H9N2亚型和鸡毒霉形体HS株主要抗原性基因的克隆与表达研究
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摘要
禽流感病毒(AIV)和鸡毒霉形体(MG)是危害世界养禽业的主要病原,对世界养禽业造成了巨大的经济损失。近年来,禽流感在我国开始流行,特别是2004年初高致病性禽流感在我国和部分亚洲国家爆发流行,不仅造成了养禽业的巨大经济损失,而且导致人类的感染和死亡,引起了世界各国政府和普通民众的高度重视。另外,鸡毒霉形体是所有禽类呼吸道感染中的最基础的病原之一,在鸡群中感染普遍,血清学阳性率高,可经蛋传播,而且经常与其他呼吸道疾病混合感染,给畜牧业生产造成了极大的经济损失。为了养禽业的稳定发展和人类健康,必须加强这两种疾病病原的主要抗原基因的分子生物学研究,为最终控制和消灭这两种疾病打下良好的基础。
本研究首先进行了禽流感病毒核蛋白(NP)、血凝素蛋白(HA)基因的克隆与表达研究。(1):采用RT-PCR方法,以AIV A/chicken/China/HSS2004(H9N2)株为材料,获得了NP基因的全长cDNA和HA基因的部分cDNA。(2):将获得的cDNA克隆入pMD18-T载体进行序列测定,测得NP基因cDNA序列长度为1525bp;HA基因cDNA序列长度为1172bp,HA切割位点附近的氨基酸组成分别是:R-S-SR↓G,切割位点附近氨基酸残基的这种组成符合低致病力毒株的分子特征。(3):与Genebank中基因序列的同源性比较分析得知,NP基因序列高度的保守性,具有型特异性,同亚型病毒间达97%,不同亚型病毒间亦达90%以上;HA基因与国内亚型病毒分离株间的同源性高达99%左右。说明国内的H9N2可能源自同一毒株。(4):构建了重组表达载体pKG-NP和pKG-HA,并在BL21 codon plus和BL21(DE3)中表达,结果pKG-NP在BL21 codon plus中获得了高效表达。SDS-PAGE和Western-blot分析表明,融合蛋白GST-NP大小约为84kD,约占菌体总蛋白的20%,能与鸡抗AIV抗体发生明显的抗原抗体反应。(5):利用十二烷基肌酸钠(SKL)方法纯化了GST-NP包涵体,并经过包涵体变性、复性、透析和包埋过程,获得了较高纯度的重组NP。
本研究在前期研究工作的基础上,对一个来自鸡毒霉形体的、含有4个pMGA基因的重组质粒(MgW17)进行了亚克隆,得到了一个完整的pMGA基因(H-pMGA1.2),为更好地研究该基因产物的活性及高效表达,我们剔除了信号肽序列以及前端的跨膜疏水部分及膜内部分;将该DNA片段插入GST融合表达载体中并在BL21中进行了表达,表达产物大小约52kD,该产物为
Avian influenza virus (AIV) and Mycoplasma gallisepticum (MG) are two of the most important causes of respiratory diseases that damage the poultry industry heavily and bring huge financial loss. In recent years, AIV have been epidemic in China, especially high pathogenic AIV burned out in China and some other countries in Asia at the beginning of 2004. Both the govements of the world and the ordinary people were focusing on the burst of AIV, not only because of the huge damage to poultry industry in economy but because of the infection and death of the human being. Addionally, MG is one of the most common causes of the respiratory diseases. Its infection rate is very high, often high to more than 50%. Furthermore, MG can be spread by eggs and frequently follow by the secondly affection and leads to heavy damage in poultry. In order to enhance the products of poultry industry and protect the health of people, it is imperative to investigate the traits of the major antigenic gene in molecular biology and tend to contribute to the study of early diagnose and control of the two diseases.In this paper, studies on cloning and expressing of major antigenic genes of avian influenza virus A/chicken/China/HSS2004 (H9N2) strain and MG-HS strain was carried out. First, nucleoprotein (NP) and hemagglutinin (HA) gene was cloned and expressed in E.coli.The methods and results reported as following. (1) NP gene cDNA and HA gene partial cDNA were amplified with the method of reverse transcription polymerization chain reaction (RT-PCR). The cDNAs were cloned into pMD-18T vector and screened positive clones to sequence. (2) The full-length cDNA of NP gene was 1525bp and shared high homology with that of the same avian subtype (97%) or other subtype (more than 90%). The cDNA of HA gene obtained was 1172bp and shared nearly 99% homology with that of the same avian subtype isolated in China, which suggested that H9N2 in China come from the same strain. Analyzing the split site of HA, structure of R-S-S-R ↓ G located near the site which was low pathogen AIV feature. (3) Recombinant plasmid pKG-NP and pKG-HA were constructed to express NP gene and HA gene in E.coli. After Inducing by IPTG, a high efficiency expression of fusion protein GST-NP derived from pKG-NP was obtained in BL21 codon plus. The level of expression was high up to 20%
of the total cell proteins of E.coli. Molecular weight of expressed products is approximately 84kD by SDS-PAGE analysis. Results of Western-blotting showed that GST-NP could react to the chicken antiserum against AIV with high immunoiogical competence. While pKG-HA failed to express in BL21 codon plus or BL21 (DE3). (4) Inclusion body of recombinant NP was purified by N-larcosine Na salt (SKL). Through denaturing, annealing, permeating and concentrating, purified recombinant NP was obtained.Second, based on the work of Changjiang Weng and Qingchun Shen, pMGA1.2, one pMGA gene in MgW17, was subcloned by polymerase chain reaction (PCR). The cloned DNA was cloned into prokaryotic vector pGEX-KG and expressed in E.coli BL21 (DE3).The product was near 55kD comprised of 26 kD GST and 243 remainder amino acid. Western-blot suggested the product react with serum of chicken anti MG.Based on these work, three "TGA" were same-sense mutated into "TGG" and got four fragments I , II, Illand IV, the molecule weight were 730bp, 174bp> 387bp and 546bp respectively. After digestion and ligatation, pMGA1.2 including three "TGG" were inserted into pGEX-KG and expressed in E.coli BL21 (DE3). Products approximately 92 kD was detected by SDS-PAGE analysis. The level of expression was high up to 25% of the total cell proteins of E.coli. Western-blot revealed that fusion protein could react with the antibody of Mycoplasma gallisepticum and showed high immunoiogical activity.Additionally, fragments Eland ITJ+IV products were obtained too, which were 39kD and 60kD and showed immunoiogical activity. These studies would provide a useful base for serological diagnosis and vaccine study and prevention of MG infection.In this study, NP gene and HA gene were obtained by RT-PCR. NP gene was expressed in E.coli at high level. At the same time, pMGA1.2 gene was subcloned and in which three TGA encoding Thr were same-sense mutated. The modified pMGA1.2 gene was expressed in E.coli at high level. Recombinant NP and recombinant pMGA provided the patient antigen in high purity and lower cost in the detection of antibody and early rapid diagnose of AIV and MG infection and make it feasible to study on the gene engineer vaccines.
本研究首先进行了禽流感病毒核蛋白(NP)、血凝素蛋白(HA)基因的克隆与表达研究。(1):采用RT-PCR方法,以AIV A/chicken/China/HSS2004(H9N2)株为材料,获得了NP基因的全长cDNA和HA基因的部分cDNA。(2):将获得的cDNA克隆入pMD18-T载体进行序列测定,测得NP基因cDNA序列长度为1525bp;HA基因cDNA序列长度为1172bp,HA切割位点附近的氨基酸组成分别是:R-S-SR↓G,切割位点附近氨基酸残基的这种组成符合低致病力毒株的分子特征。(3):与Genebank中基因序列的同源性比较分析得知,NP基因序列高度的保守性,具有型特异性,同亚型病毒间达97%,不同亚型病毒间亦达90%以上;HA基因与国内亚型病毒分离株间的同源性高达99%左右。说明国内的H9N2可能源自同一毒株。(4):构建了重组表达载体pKG-NP和pKG-HA,并在BL21 codon plus和BL21(DE3)中表达,结果pKG-NP在BL21 codon plus中获得了高效表达。SDS-PAGE和Western-blot分析表明,融合蛋白GST-NP大小约为84kD,约占菌体总蛋白的20%,能与鸡抗AIV抗体发生明显的抗原抗体反应。(5):利用十二烷基肌酸钠(SKL)方法纯化了GST-NP包涵体,并经过包涵体变性、复性、透析和包埋过程,获得了较高纯度的重组NP。
本研究在前期研究工作的基础上,对一个来自鸡毒霉形体的、含有4个pMGA基因的重组质粒(MgW17)进行了亚克隆,得到了一个完整的pMGA基因(H-pMGA1.2),为更好地研究该基因产物的活性及高效表达,我们剔除了信号肽序列以及前端的跨膜疏水部分及膜内部分;将该DNA片段插入GST融合表达载体中并在BL21中进行了表达,表达产物大小约52kD,该产物为
Avian influenza virus (AIV) and Mycoplasma gallisepticum (MG) are two of the most important causes of respiratory diseases that damage the poultry industry heavily and bring huge financial loss. In recent years, AIV have been epidemic in China, especially high pathogenic AIV burned out in China and some other countries in Asia at the beginning of 2004. Both the govements of the world and the ordinary people were focusing on the burst of AIV, not only because of the huge damage to poultry industry in economy but because of the infection and death of the human being. Addionally, MG is one of the most common causes of the respiratory diseases. Its infection rate is very high, often high to more than 50%. Furthermore, MG can be spread by eggs and frequently follow by the secondly affection and leads to heavy damage in poultry. In order to enhance the products of poultry industry and protect the health of people, it is imperative to investigate the traits of the major antigenic gene in molecular biology and tend to contribute to the study of early diagnose and control of the two diseases.In this paper, studies on cloning and expressing of major antigenic genes of avian influenza virus A/chicken/China/HSS2004 (H9N2) strain and MG-HS strain was carried out. First, nucleoprotein (NP) and hemagglutinin (HA) gene was cloned and expressed in E.coli.The methods and results reported as following. (1) NP gene cDNA and HA gene partial cDNA were amplified with the method of reverse transcription polymerization chain reaction (RT-PCR). The cDNAs were cloned into pMD-18T vector and screened positive clones to sequence. (2) The full-length cDNA of NP gene was 1525bp and shared high homology with that of the same avian subtype (97%) or other subtype (more than 90%). The cDNA of HA gene obtained was 1172bp and shared nearly 99% homology with that of the same avian subtype isolated in China, which suggested that H9N2 in China come from the same strain. Analyzing the split site of HA, structure of R-S-S-R ↓ G located near the site which was low pathogen AIV feature. (3) Recombinant plasmid pKG-NP and pKG-HA were constructed to express NP gene and HA gene in E.coli. After Inducing by IPTG, a high efficiency expression of fusion protein GST-NP derived from pKG-NP was obtained in BL21 codon plus. The level of expression was high up to 20%
of the total cell proteins of E.coli. Molecular weight of expressed products is approximately 84kD by SDS-PAGE analysis. Results of Western-blotting showed that GST-NP could react to the chicken antiserum against AIV with high immunoiogical competence. While pKG-HA failed to express in BL21 codon plus or BL21 (DE3). (4) Inclusion body of recombinant NP was purified by N-larcosine Na salt (SKL). Through denaturing, annealing, permeating and concentrating, purified recombinant NP was obtained.Second, based on the work of Changjiang Weng and Qingchun Shen, pMGA1.2, one pMGA gene in MgW17, was subcloned by polymerase chain reaction (PCR). The cloned DNA was cloned into prokaryotic vector pGEX-KG and expressed in E.coli BL21 (DE3).The product was near 55kD comprised of 26 kD GST and 243 remainder amino acid. Western-blot suggested the product react with serum of chicken anti MG.Based on these work, three "TGA" were same-sense mutated into "TGG" and got four fragments I , II, Illand IV, the molecule weight were 730bp, 174bp> 387bp and 546bp respectively. After digestion and ligatation, pMGA1.2 including three "TGG" were inserted into pGEX-KG and expressed in E.coli BL21 (DE3). Products approximately 92 kD was detected by SDS-PAGE analysis. The level of expression was high up to 25% of the total cell proteins of E.coli. Western-blot revealed that fusion protein could react with the antibody of Mycoplasma gallisepticum and showed high immunoiogical activity.Additionally, fragments Eland ITJ+IV products were obtained too, which were 39kD and 60kD and showed immunoiogical activity. These studies would provide a useful base for serological diagnosis and vaccine study and prevention of MG infection.In this study, NP gene and HA gene were obtained by RT-PCR. NP gene was expressed in E.coli at high level. At the same time, pMGA1.2 gene was subcloned and in which three TGA encoding Thr were same-sense mutated. The modified pMGA1.2 gene was expressed in E.coli at high level. Recombinant NP and recombinant pMGA provided the patient antigen in high purity and lower cost in the detection of antibody and early rapid diagnose of AIV and MG infection and make it feasible to study on the gene engineer vaccines.
引文
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