丙型肝炎病毒高变区1高反应性多肽筛选、表达及其特性研究
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摘要
高变区1（HVR1）是位于丙型肝炎病毒（HCV）被膜蛋白E2氨基端的高度异质性的一段序列，被认为是宿主体液免疫中和抗体的重要靶位，但其株特异性可导致部分病毒逃避宿主的免疫识别和感染的持续。虽然如此，HVR1序列中存在较保守的位点，通过合适的HVR1免疫程序，仍可能诱生出保护性抗体。
本研究对来自中国不同地区的HCV HVR1进行了序列特性分析，在综合考虑地域因素和基因型的差别、亲水性和免疫原性的基础上，首先在大肠杆菌中表达了四种DHFR-HVR1融合蛋白，检测了它们与HCV感染者血清反应性，其中SH1b的反应率最高，为66.67%；初步分析提示患者免疫反应的强度可能与干扰素疗效有关。所得融合蛋白纯化后免疫家兔获得了相应的抗体。将其中三个HVR1片段分别连接至乙型肝炎病毒表面抗原片段的羧端，表达了HBs-HVR1融合蛋白。
为进一步提高HVR1序列的交叉反应性，采用DNA shuffling的方法体外重组31株从丙肝患者血清中获得的HVR1 cDNA自然序列，构建HVR1模拟序列噬菌体展示文库，通过抗HVR1多克隆抗体的亲和筛选， 其反应性从46.7%-66.7% 提高到 53.3%-80%，二株反应性最高的HVR1模拟肽为13号（80%）和23号（76.7%），提示它们可能是重要的免疫原性肽段，其序列与共识序列高度同源可能是导致其广泛反应性的原因。对模拟肽的免疫原性及保护性将在今后作进一步的研究。
总之，本研究在原核及真核系统中成功地表达了所选择的HVR1序列，并对其部分特性进行了分析；同时采用DNA shuffling和噬菌体展示技术，构建了HVR1模拟肽库，筛选得到了高反应性模拟肽。与目前国际上广泛采用的合成肽研究方式相比，费用便宜，变异类型覆盖面广，交叉反应性高，可望成为抗HCV感染的候选免疫原。
Hypervariable region 1 (HVR1) is a highly heterogeneous sequence located at the N terminus of the second envelope protein (E2) of hepatitis C virus (HCV) and has been proved to be the target of neutralizing antibodies but with isolate specificity, which involved in the escape from host immune recognition. However, some studies revealed that the protective antibodies might be obtained through conserved sites of HVR1 by convenient immunization process.
Based on the variability analysis of HVR1 sequences, gene fragments of HVR1 of four HCV strains (three genotype 1b and one genotype 2a) were selected and amplified from pGEMT-E plasmids and sub-cloned into pQE40 vectors respectively to construct recombinant expression plasmids which expressed HVR1 fused downstream to DHFR in Escherichia coli. The reactivity of the four fusion proteins was detected in serum samples of patients with chronic hepatitis C. Among them, SH1b was the most reactive one (66.67%). The results revealed that the intensity and/or quality of the immune response against HCV might be a critical factor determining the response to interferon treatment. Meanwhile, the purified DHFR- HVR1 proteins were used to immunize rabbits and antibodies anti-HVR1 were obtained. Three of the four HVR1 sequences were also expressed as fusion proteins with HBsAg in mammalian cells.
To further improve the cross-reactivity of HVR1 sequences, thirty-one HVR1 cDNA fragments were digested by DNase Ⅰ into a pool of random fragments and reassembled by repeated cycles of annealing in the presence of DNA polymerase to their original size. The shuffled HVR1 were then inserted into the gene III phagemid vector pCANTAB-5E and displayed on the surface of the phage. After four rounds of biopanning against anti-HVR1, the reactivity of eight selected clones to this panel of sera were from 46.7%-66.7% to 53.3%-80%. The two highest reactive phages (phage 13,
80% and phage 23, 76%) had the similar amino acid sequences with the consensus sequences defined by Puntoriero et al, which might play a particular role in determining the frequency of reactivity.
In conclusion, this study has successfully expressed four selected HVR1 sequences in prokaryotic and mammalian system and evaluated their characteristics. Meanwhile, DNA shuffling combined with phage display technology was effectively utilized to identify broadly cross-reactive mimotopes recognized by human polyclonal antibodies. Compared with synthetic peptides widely used in other studies, these techniques, costing much less and covering more variants, had got the broadly reactive sequences. Mimotope 13 and 23 appeared to be immunologically most reactive and could be immunogen candidate. Efforts are now underway to identify their neutralizing antibodies by immunization of animals.
本研究对来自中国不同地区的HCV HVR1进行了序列特性分析，在综合考虑地域因素和基因型的差别、亲水性和免疫原性的基础上，首先在大肠杆菌中表达了四种DHFR-HVR1融合蛋白，检测了它们与HCV感染者血清反应性，其中SH1b的反应率最高，为66.67%；初步分析提示患者免疫反应的强度可能与干扰素疗效有关。所得融合蛋白纯化后免疫家兔获得了相应的抗体。将其中三个HVR1片段分别连接至乙型肝炎病毒表面抗原片段的羧端，表达了HBs-HVR1融合蛋白。
为进一步提高HVR1序列的交叉反应性，采用DNA shuffling的方法体外重组31株从丙肝患者血清中获得的HVR1 cDNA自然序列，构建HVR1模拟序列噬菌体展示文库，通过抗HVR1多克隆抗体的亲和筛选， 其反应性从46.7%-66.7% 提高到 53.3%-80%，二株反应性最高的HVR1模拟肽为13号（80%）和23号（76.7%），提示它们可能是重要的免疫原性肽段，其序列与共识序列高度同源可能是导致其广泛反应性的原因。对模拟肽的免疫原性及保护性将在今后作进一步的研究。
总之，本研究在原核及真核系统中成功地表达了所选择的HVR1序列，并对其部分特性进行了分析；同时采用DNA shuffling和噬菌体展示技术，构建了HVR1模拟肽库，筛选得到了高反应性模拟肽。与目前国际上广泛采用的合成肽研究方式相比，费用便宜，变异类型覆盖面广，交叉反应性高，可望成为抗HCV感染的候选免疫原。
Hypervariable region 1 (HVR1) is a highly heterogeneous sequence located at the N terminus of the second envelope protein (E2) of hepatitis C virus (HCV) and has been proved to be the target of neutralizing antibodies but with isolate specificity, which involved in the escape from host immune recognition. However, some studies revealed that the protective antibodies might be obtained through conserved sites of HVR1 by convenient immunization process.
Based on the variability analysis of HVR1 sequences, gene fragments of HVR1 of four HCV strains (three genotype 1b and one genotype 2a) were selected and amplified from pGEMT-E plasmids and sub-cloned into pQE40 vectors respectively to construct recombinant expression plasmids which expressed HVR1 fused downstream to DHFR in Escherichia coli. The reactivity of the four fusion proteins was detected in serum samples of patients with chronic hepatitis C. Among them, SH1b was the most reactive one (66.67%). The results revealed that the intensity and/or quality of the immune response against HCV might be a critical factor determining the response to interferon treatment. Meanwhile, the purified DHFR- HVR1 proteins were used to immunize rabbits and antibodies anti-HVR1 were obtained. Three of the four HVR1 sequences were also expressed as fusion proteins with HBsAg in mammalian cells.
To further improve the cross-reactivity of HVR1 sequences, thirty-one HVR1 cDNA fragments were digested by DNase Ⅰ into a pool of random fragments and reassembled by repeated cycles of annealing in the presence of DNA polymerase to their original size. The shuffled HVR1 were then inserted into the gene III phagemid vector pCANTAB-5E and displayed on the surface of the phage. After four rounds of biopanning against anti-HVR1, the reactivity of eight selected clones to this panel of sera were from 46.7%-66.7% to 53.3%-80%. The two highest reactive phages (phage 13,
80% and phage 23, 76%) had the similar amino acid sequences with the consensus sequences defined by Puntoriero et al, which might play a particular role in determining the frequency of reactivity.
In conclusion, this study has successfully expressed four selected HVR1 sequences in prokaryotic and mammalian system and evaluated their characteristics. Meanwhile, DNA shuffling combined with phage display technology was effectively utilized to identify broadly cross-reactive mimotopes recognized by human polyclonal antibodies. Compared with synthetic peptides widely used in other studies, these techniques, costing much less and covering more variants, had got the broadly reactive sequences. Mimotope 13 and 23 appeared to be immunologically most reactive and could be immunogen candidate. Efforts are now underway to identify their neutralizing antibodies by immunization of animals.
引文
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Zhang XX，Zhang SY, Liu J, Lu ZM, Wang Y. Expression of hepatitis C virus hypervariable region 1 and its clinical significance. World J gastroenterology 2003;
95. accepted
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94. Cai Q, Zhang XX, Tian LF, Yuan M, Jin GD, Lu ZM. Variant analysis and immunogenicity prediction of envelope gene of HCV strains from China. J Med Virol 2002; 67: 490-500.
Zhang XX，Zhang SY, Liu J, Lu ZM, Wang Y. Expression of hepatitis C virus hypervariable region 1 and its clinical significance. World J gastroenterology 2003;
95. accepted
96. Global surveillance and control of hepatitis C virus. Report of a WHO consultation organized in collaboration with the Viral Hepatitis Prevention Board. Antwerp, Belgium. J Viral Hepat 1999; 6: 35-47.