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目的 研究HAV 3a(位于 14 0 3～ 14 5 6aa)与 3d(位于 1719～ 176 4aa)融合蛋白在原核系统中的表达 ,并探讨该重组蛋白的抗原活性及其应用价值。方法 采用常规PCR方法 ,从克隆有HAVHM175株全长cDNA基因的克隆载体pHAV16H1上扩增出目的基因 ,以pET 30a作为表达载体 ,构建重组表达质粒pET 3ad。该重组质粒在大肠埃希菌BL2 1(DE3)中经IPTG诱导表达。采用镍金属柱螯和亲和层析纯化的方法对重组蛋白进行纯化。以Westernblot和间接ELISA的方法检测重组蛋白的抗原活性。结果 重组质粒pET 3ad经双酶切 (NcoⅠ /HindⅢ )鉴定和序列测定证实构建成功。在大肠埃希菌BL2 1(DE3)中诱导表达有一相对分子质量为 180 0 0的重组蛋白P3ad ,该蛋白经亲和层析获得了良好的纯化效果。Westernblot结果显示在相对分子质量为 180 0 0处有一特异的免疫印迹条带 ,表明重组蛋白P3ad具有特异的抗原活性 ;间接ELISA方法进一步证实了该蛋白的抗原性。结论 采用常规的克隆操作方法构建了重组表达质粒pET 3ad ,其表达量为 2 0 %。表达产物具有良好的抗原性 ,有望应用于急性HAV感染的诊断和区分活病毒的感染及灭活疫苗的免疫。
Objective To investigate the expression of the fusion protein HAV 3a (located at 143 0 to 146 6a4) and 3d (located at 1719 176 176a) in prokaryotic system, and to explore the antigenic activity of the recombinant protein and its application value. Methods The recombinant plasmid pET 3ad was constructed by cloning the full length cDNA of HAVHM175 from pHAV16H1 cloned by conventional PCR. PET 30a was constructed by using pET 30a as the expression vector. The recombinant plasmid was induced by IPTG in Escherichia coli BL21 (DE3). Recombinant proteins were purified by nickel metal chelating and affinity chromatography. The antigenic activity of the recombinant protein was detected by Western blot and indirect ELISA. Results The recombinant plasmid pET 3ad was successfully identified by NcoⅠ / Hind Ⅲ sequencing and sequence analysis. A recombinant protein P3ad was expressed in Escherichia coli BL21 (DE3) with a relative molecular mass of 180,000. The protein was purified by affinity chromatography. Western blot showed that there was a specific immunoblotting band at a molecular weight of 180,000, indicating that the recombinant protein P3ad has specific antigenic activity. The indirect ELISA method further confirmed the antigenicity of the protein. Conclusion The recombinant plasmid pET 3ad was constructed by a routine cloning method and its expression level was 20%. The expression product has good antigenicity and is expected to be used in the diagnosis of acute HAV infection and to distinguish the infection of live virus and the immunity of inactivated vaccine.