戊型肝炎病毒ORF2抗原的制备及其免疫原性评价
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摘要
目的制备戊型肝炎病毒(hepatitis E virus,HEV)ORF2抗原,并对其免疫原性进行分析。方法通过基因合成获得HEV ORF2目的基因,构建重组杆状病毒,并在sf9昆虫细胞中优化表达;采用超声、硫酸铵沉淀及层析纯化HEV ORF2蛋白,免疫小鼠分析其免疫原性。结果制备的重组杆状病毒滴度为1×108 PFU/m L;重组蛋白表达条件MOI为1,收获时间为5 d时,表达量最高为20 mg/L;通过纯化获得HEV ORF2蛋白纯度> 95%;其疫苗组血清特异性抗体效价与PBS对照组比较差异有统计学意义(P <0. 05)。结论成功表达并纯化了HEV ORF2蛋白,该纯化方法可获得纯度较高的目的蛋白,且制备的HEV ORF2蛋白具有很好的免疫原性,可用于制备和开发预防性疫苗。
Objective To prepare the ORF2 antigen of hepatitis E virus(HEV)and evaluate its immunogenicity. Methods The gene encoding HEV ORF2 protein was synthesized and expressed in sf9 cells by recombinant baculovius. The expressed HEV ORF2 protein was purified by ultrasonication,precipitation with ammonium sulphate and chromatography,of which the immunogenicity was evaluated in mice. Results The prepared recombinant baculovirus reached a titer of 1 × 108 PFU/mL. The optimal MOI for expression was 1,while the optimal time for harvest was 5 d. The expression level at the optimal condition was 20 mg/L at most. The purified HEV ORF2 protein reached a purity of more than 95%.The specific antibody titer induced by the protein in sera was significantly higher than that by PBS as control(P < 0. 05).Conclusion HEV ORF2 protein was successfully expressed and purified. The purified HEV ORF2 protein reached a high purity,which showed good immunogenicity and might be used for preparation and development of preventive vaccine.
Objective To prepare the ORF2 antigen of hepatitis E virus(HEV)and evaluate its immunogenicity. Methods The gene encoding HEV ORF2 protein was synthesized and expressed in sf9 cells by recombinant baculovius. The expressed HEV ORF2 protein was purified by ultrasonication,precipitation with ammonium sulphate and chromatography,of which the immunogenicity was evaluated in mice. Results The prepared recombinant baculovirus reached a titer of 1 × 108 PFU/mL. The optimal MOI for expression was 1,while the optimal time for harvest was 5 d. The expression level at the optimal condition was 20 mg/L at most. The purified HEV ORF2 protein reached a purity of more than 95%.The specific antibody titer induced by the protein in sera was significantly higher than that by PBS as control(P < 0. 05).Conclusion HEV ORF2 protein was successfully expressed and purified. The purified HEV ORF2 protein reached a high purity,which showed good immunogenicity and might be used for preparation and development of preventive vaccine.
引文
[1] CAO Y F, TAO H, HU YM, et al. A phase. 1 randomized openlabel clinical study to evaluate the safety and tolerability of a novel recombinant hepatitis E vaccine[J]. Vaccine,2017,35(37):5073-5080.
[2] JIN H, ZHAO Y, ZHANG X, et al. Case-fatality risk of pregnant women with acute viral hepatitis type E:a systematic review and meta-analysis[J]. Epidemiol Infect, 2016,144(10):2098-2106.
[3] CHAUDHRY S A, KOREN G. Hepatitis A infection during pregnancy[J]. Can Family Physician Med, 2015, 61(11):963-964.
[4] GUERRA J, KAMPA K C, MORSOLETTO D G B, et al. Hepatitis E:a literature review[J]. J Clin Translat Hepatol, 2017,5(4):376-383.
[5] MURALI A R, KOTWAL V, CHAWLA S. Chronic hepatitis E:A brief review[J]. World J Hepatol, 2015, 7(19):2194-2201.
[6] SRIDHAR S,LO S K,XING F,et al. Clinical characteristics and molecular epidemiology of hepatitis E in Shenzhen, China:a shift toward foodborne transmission of hepatitis E virus infection[j]. Emerg Microb&Infect, 2017, 6(12):e115.
[7] REN X, WU P, WANG L, et al. Changing epidemiology of hepatitis A and hepatitis E viruses in China, 1990-2014[J].Emerg Infect Dis, 2017, 23(2):276-279.
[8] MENG X J. Zoonotic and foodborne transmission of hepatitis E virus[J]. Seminars Liver Dis, 2013, 33(1):41-49.
[9] YUGO D M, MENG X J. Hepatitis E virus:foodborne, waterborne and zoonotic transmission[J]. Int J Environ Res Public Health, 2013, 10(10):4507-4533.
[10] WANG L, LIU L, WEI Y, et al. Clinical and virological profiling of sporadic hepatitis E virus infection in China[J]. J Infect, 2016, 73(3):271-279.
[11] LI T C, TAKEDA N, MIYAMURA T, et al. Essential elements of the capsid protein for self-assembly into empty virus-like particles of hepatitis E virus[J]. J Virol, 2005, 79(20):12999-13006.
[12] LI T C, KATAOKA M, TAKAHASHI K, et al. Generation of hepatitis E virus-like particles of two new genotypes G5 and G6and comparison of antigenic properties with those of known genotypes[J]. Veter Microbiol, 2015, 178(1-2):150-157.
[13] SHIM A R, LI T C, SENDAI Y, et al. Production of hepatitis E virus-like particles presenting multiple foreign epitopes by coinfection of recombinant baculoviruses[J]. Sci Rep,2016,6:21638.
[2] JIN H, ZHAO Y, ZHANG X, et al. Case-fatality risk of pregnant women with acute viral hepatitis type E:a systematic review and meta-analysis[J]. Epidemiol Infect, 2016,144(10):2098-2106.
[3] CHAUDHRY S A, KOREN G. Hepatitis A infection during pregnancy[J]. Can Family Physician Med, 2015, 61(11):963-964.
[4] GUERRA J, KAMPA K C, MORSOLETTO D G B, et al. Hepatitis E:a literature review[J]. J Clin Translat Hepatol, 2017,5(4):376-383.
[5] MURALI A R, KOTWAL V, CHAWLA S. Chronic hepatitis E:A brief review[J]. World J Hepatol, 2015, 7(19):2194-2201.
[6] SRIDHAR S,LO S K,XING F,et al. Clinical characteristics and molecular epidemiology of hepatitis E in Shenzhen, China:a shift toward foodborne transmission of hepatitis E virus infection[j]. Emerg Microb&Infect, 2017, 6(12):e115.
[7] REN X, WU P, WANG L, et al. Changing epidemiology of hepatitis A and hepatitis E viruses in China, 1990-2014[J].Emerg Infect Dis, 2017, 23(2):276-279.
[8] MENG X J. Zoonotic and foodborne transmission of hepatitis E virus[J]. Seminars Liver Dis, 2013, 33(1):41-49.
[9] YUGO D M, MENG X J. Hepatitis E virus:foodborne, waterborne and zoonotic transmission[J]. Int J Environ Res Public Health, 2013, 10(10):4507-4533.
[10] WANG L, LIU L, WEI Y, et al. Clinical and virological profiling of sporadic hepatitis E virus infection in China[J]. J Infect, 2016, 73(3):271-279.
[11] LI T C, TAKEDA N, MIYAMURA T, et al. Essential elements of the capsid protein for self-assembly into empty virus-like particles of hepatitis E virus[J]. J Virol, 2005, 79(20):12999-13006.
[12] LI T C, KATAOKA M, TAKAHASHI K, et al. Generation of hepatitis E virus-like particles of two new genotypes G5 and G6and comparison of antigenic properties with those of known genotypes[J]. Veter Microbiol, 2015, 178(1-2):150-157.
[13] SHIM A R, LI T C, SENDAI Y, et al. Production of hepatitis E virus-like particles presenting multiple foreign epitopes by coinfection of recombinant baculoviruses[J]. Sci Rep,2016,6:21638.