华支睾吸虫NOSIP基因的克隆表达及免疫学鉴定

Cloning, expression, and immunological characterization of a nitric oxide synthase-interacting protein from Clonorchis sinensis

目的对华支睾吸虫一氧化氮合酶相互作用蛋白(CsNOSIP)进行克隆和原核表达,初步了解其重组产物的免疫学功能。 方法从华支睾吸虫基因文库中获得CsNOSIP的全长cDNA并克隆至原核表达质粒pET-30a(+)中,诱导表达后用亲和层析柱进行纯化,用纯化的rCsNOSIP及rCsNOSIP蛋白免疫BALB/c小鼠以获得rCsNOSIP免疫血清,采用Western blot鉴定重组蛋白CsNOSIP的表达及其反应原性;采用ELISA检测rCsNOSIP免疫小鼠血清特异性抗体亚类水平。 结果CsNOSIP基因的开放阅读框(ORF)包含867bp,编码288个氨基酸,PCR、双酶切及DNA测序表明pET-30a(+)-CsNOSIP重组质粒构建成功。SDS-PAGE检测目的蛋白在大肠埃希菌BL21/DE3中获得高效表达,表达产物相对分子质量为35×103;经亲和层析法获得高纯度的重组蛋白,该蛋白可被His单抗、CsNOSIP免疫小鼠血清、感染华支睾吸虫小鼠血清及ESP免疫血清识别,重组蛋白免疫血清能识别CsESP;ELISA检测显示rCsNOSIP免疫小鼠血清特异抗体滴度为1∶25 600,以IgG1水平较高。 结论CsNOSIP可在原核表达系统中呈现高效可溶性表达,且具有抗原性,是华支睾吸虫分泌排泄抗原(CsESP)之一,免疫小鼠后可获得高滴度的特异性抗体,抗体亚类以IgG1为主,为该蛋白的功能研究奠定了基础。

Objectives To clone and express the gene encoding a nitric oxide synthase-interacting protein(NOSIP) fromClonorchis sinensis and to study the antigenicity of a recombinant protein. Methods The published genome of C. sinensis was used to investigate a gene encoding a NOSIP of C.sinensis(CsNOSIP).The open reading frame of CsNOSIP was amplified using PCR,cloned into the prokaryotic expression vector pET-30a(+),and then expressed in Escherichia coli BL21.The recombinant protein was purified using NI-DNA affinity chromatography and detected using SDS-PAGE. Recombinant CsNOSIP(rCsNOSIP)was used to produce polyclonal antibodies in BALB/c mice.The immune reactivity and antigenicity of CsNOSIP were analyzed using Western blotting.Serum specificity and antibody titers were determined using an enzyme-linked immunosorbent assay(ELISA). Results Results indicated that the complete cDNA sequence of CsNOSIP is 867bp in length and that it encodes 289amino acids.The theoretical molecular weight of CsNOSIP is 35 000 Mr.PCR,double enzyme digestion,and DNA sequencing indicated that the recombinant plasmid pET-30a(+)- CsNOSIP was successfully constructed.rCsNOSIP was efficiently expressed and purified fromEscherichia coli BL21,and the relative molecular weight of the product was about 35 000Mr.The recombinant protein was obtained via affinity chromatography. Moreover,Western blotting indicated that rCsNOSIP was recognized by mouse anti-His-tag monoclonal antibodies, serum from BALB/c mice immunized with the rCsNOSIP protein,serum from BALB/c mice infected with C.sinensis, and serum from BALB/c mice immunized with excretory secretory products(ESPs).Furthermore,ELISA indicated that the serum specific IgG antibody titer was 1:25 600after BALB/c mice were immunized with rCsNOSIP,resulting in higher IgG1levels. Conclusion rCsNOSIP is highly expressed in E.coli as a soluble protein,and it retains its antigenicity. CsNOSIP was confirmed to be a component of CsESPs.Analysis of the antibody subclass and antibody titer indicated that BALB/c mice subcutaneously immunized with rCsNOSIP had significantly elevated serum IgG1levels and highly specific antibodies.These findings have laid a solid foundation for subsequent study of the function of this protein.