HAV 3a与3d融合蛋白在原核系统中的表达及其抗原活性的研究

目的 研究HAV 3a(位于1403-1456aa)与3d(位于1719-1764aa)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HAV HM175株全长cDNA基因的克隆载体pHAVl6H1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BL21(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Western blot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(Nco I／Hind Ⅲ)鉴定和序列测定证实构建成功。在大肠埃希菌BL21(DE3)中诱导表达有一相对分子质量为18000的重组蛋白P3ad，该蛋白经亲和层析获得了良好的纯化效果。Western blot结果显示在相对分子质量为18000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20％。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。

Expression of the 3a and 3d fusion protein of hepatitis A virus in prokaryotic cell and antigenicity analysis

OBJECTIVE: To fusionaly express the HAV 3a (located in 1403-1456 aa) and 3d located in 1719-1764 aa cDNA gene fragments in prokaryotic system; to investigate the antigenicity and application of recombinant protein. METHODS: By using PCR technique, 3a and 3d gene fragments were cloned. Choosing pET-30a as the expressive vector, the recombinant plasmid Pet-3ad was constructed and pET-3ad was expressed in Escherichia coli after inducing by IPTG. By affinity chromatography, purified recombinant protein was obtained. By using Western blot analysis and indirect ELISA to detect its antigenic activity. RESULTS: Recombinant plasmid pET-3ad was proved to construct successfully by enzyme-digestion and sequence measurement. Recombinant protein P3ad(18,000) was obtained in BL21(DE3) and purified after Ni+ affinity chromatography. Western blot analysis and indirect ELISA showed that P3ad had specific antigenicity. CONCLUSIONS: Recombinant plasmid pET-3ad was proved to construct successfully by enzyme-digestion (Nco I/Hind III) and sequence measurement. Recombinant protein P3ad(18,000) was obtained in BL21(DE3) and purified after Ni+ affinity chromatography. Specific immunoblotting appeared at 18,000 by western blot analysis, which showed the recombinant protein P3ad had specific antigenicity, indirect ELISA further proved its antigenicity.