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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 and purify the fusion protein of hepatitis B virus (HBcAg) (1 ~ 155) and preS1 antigen (PreS1) (3 ~ 55) in prokaryotic cells and analyze its immunogenicity. Methods The HBV DNA was extracted from the serum of patients with HBeAg-positive chronic hepatitis B, and the partial HBcAg and preS1 gene fragments and the fusion gene CS1 were amplified by PCR. The CS1 gene was subcloned into prokaryotic expression vector pET-32a (+), The recombinant prokaryotic expression vector pET-CS1 was constructed and transformed into E. coli BL21 (DE3) for expression under induction of IPTG. The expressed product was purified and analyzed by SDS-PAGE, HPLC and Western blot. The purified fusion protein was immunized with BALB / c mice. Anti-HBc level was detected by competitive inhibition method. Anti-PreS1 level was detected by indirect ELISA. Antibody subclasses were detected by ELISA. The cellular immunity was evaluated by ELISPOT. Results The recombinant prokaryotic expression plasmid pET-CS1 was confirmed by double enzyme digestion. Electron microscopy analysis showed that crude CS1 could assemble into virus-like particles (VLP) with a diameter of about 30 nm. SDS-PAGE and HPLC analysis showed that CS1 protein Purified CS1 protein could specifically bind to rabbit anti-human HBcAg-polyclonal antibody and mouse anti-human PreS1 monoclonal antibody, and could induce anti-HBc and anti-PreS1 antibodies in antibody subclasses IgG2a-based, and can induce mouse spleen cells produce HBcAg-specific IFNγ. Conclusion The fusion protein CS1 has been successfully expressed in prokaryotic cells and purified. The purity of purified CS1 is high, which can induce the body to produce high level of specific humoral and cellular immune responses.