乙型肝炎病毒核心抗原与前S1抗原融合蛋白的原核表达、纯化及其免疫原性  

Prokaryotic Expression,Purification and Immunogenicity of Fusion Protein of Hepatitis B Virus Core Antigen and PreS1 Antigen

作　　者：吴刚[1] 李计来[1] 王娟[1] 赵莉[1] 徐静[1] 

机构地区：[1]北京生物制品研究所免疫研究室,北京100024

出　　处：《中国生物制品学杂志》2011年第3期249-254,共6页Chinese Journal of Biologicals

摘　　要：目的原核表达、纯化乙型肝炎病毒(HBV)核心抗原(HBcAg)(1～155)与前S1抗原(PreS1)(3～55)融合蛋白,并分析其免疫原性。方法从HBeAg阳性慢性乙型肝炎患者血清中提取HBV DNA,以其为模板,PCR分别扩增HBcAg和preS1部分基因片段及融合基因CS1,将CS1基因亚克隆入原核表达载体pET-32a(+),构建重组原核表达质粒pET-CS1,转化大肠杆菌BL21(DE3),IPTG诱导表达,表达产物经纯化后进行SDS-PAGE、HPLC和Western blot等分析。将纯化的融合蛋白免疫BALB/c小鼠,采用竞争抑制法检测血清Anti-HBc水平,间接ELISA法检测Anti-PreS1水平,并检测抗体亚类;ELISPOT法评价其细胞免疫效果。结果重组原核表达质粒pET-CS1经双酶切证明构建正确;电镜分析表明粗纯的CS1可自行组装成病毒样颗粒(VLP),直径约为30 nm;SDS-PAGE和HPLC分析显示,CS1蛋白纯度分别为98.2%和93%;纯化的CS1蛋白可与兔抗人HBcAgr多抗和鼠抗人PreS1单抗特异结合,并能诱导小鼠产生Anti-HBc和Anti-PreS1抗体,抗体亚类以IgG2a为主,并能够诱生小鼠脾细胞产生HBcAg特异性的IFNγ。结论已成功原核表达并纯化了融合蛋白CS1,纯化的CS1纯度较高,能诱导机体产生较高水平的特异性体液免疫和细胞免疫反应。Objective To express the fusion protein of hepatitis B virus core antigen（HBcAg）（1 ～ 155） and preS1 antigen（PreS1）（3 ～ 55）,purify the expressed product and analyze its immunogenicity.Methods HBV DNA was extracted from the sera of HBeAg-positive patients with hepatitis B and used as templates for amplification of HBcAg and PreS1 genes by PCR.The fusion gene CS1 was subcloned into prokaryotic expression vector pET-32a（＋）.The constructed recombinant plasmid pET-CS1 was transformed to E.coli BL21（DE3） for expression under induction of IPTG.The expressed product was purified then identified by SDS-PAGE,HPLC and Western blot.BALB / c mice were immunized with the purified fusion protein and determined for subclasses of total Ab and titers of anti-PreS1 by indirect ELISA,and for titers of anti-HBc by competitive inhibition method,then evaluated for cellular immune effect by ELISPOT.Results Both restriction analysis and sequencing proved that recombinant plasmid pET-CS1 was constructed correctly.Electron microscopy showed that the preliminarily purified CS1 was assembled to virus-like particles at diameters of about 30 nm automatically.SDS-PAGE and HPLC showed that the purities of CS1 protein were 98.2% and 93% respectively.Purified CS1 protein was bound to anti-HBc and anti-PreS1 specifically,and induced anti-HBc and anti-PreS1 in mice,most of which were of subclass IgG2a.In addition,purified CS1 protein induced the secretion of HBcAg-specific IFNγ by murine splenic cells.Conclusion Fusion protein CS1 was successfully expressed in prokaryotic cells and purified,and the purified CS1 reached a high purity and induced high humoral and cellular immune responses.

关 键 词：肝炎核心抗原 乙型 前S1抗原 重组融合蛋白质类 体液免疫 细胞免疫 

分 类 号：R373.21[医药卫生—病原生物学] Q78[医药卫生—基础医学]