淋病奈瑟菌孔蛋白PorB的原核表达及其在疫苗血清学检测中的初步应用

Prokaryotic expression of PorB gene from neisseria gonorrhoeae and its primary application in serological detection

目的克隆并表达淋病奈瑟菌孔蛋白Por B,以重组蛋白为抗原,间接ELISA检测免疫血清的抗体水平。方法利用PCR从淋病奈瑟菌WHO株基因组中扩增出Por B的基因,克隆入原核表达载体p ET-30a中,构建出重组表达质粒p ET30aPor B,并转化大肠杆菌BL21(DE3)中,异丙基β-D-硫代半乳糖苷(IPTG)诱导表达。表达的蛋白进行SDS-PAGE分析,Western blot检测其反应原性。以纯化的重组蛋白r Por B作为检测抗原建立间接ELISA方法,用于检测淋病疫苗p VAX1-Por B免疫小鼠后的血清抗体水平。结果 PCR扩增得到淋病奈瑟菌孔蛋白Por B基因,表达的重组蛋白r Por B相对分子质量约40 k D,经Ni-NTA亲和层析纯化后的r Por B在SDS-PAGE中显示单一条带,Western blot证明纯化后的r Por B可与淋病奈瑟菌免疫血清特异性结合,具有良好的免疫反应性。间接ELISA结果表明,淋病疫苗p VAX1-Por B免疫小鼠血清Por B特异性抗体滴度为1∶200。结论成功构建了重组原核表达质粒p ET30a-Por B,Por B在大肠杆菌中获得表达。以纯化的r Por B为检测抗原,间接ELISA可以用于淋病疫苗免疫效果的评价。该研究为淋病奈瑟菌感染诊断、疫苗的研究奠定了基础。

Objective To clone the PorB protein of neisseria gonorrhoeae and use indirect ELISA to evaluate the titers of antibodies against neisseria gonorrhoeae vaccine. Methods The com- plete PorB gene from neisseria gonorrhoeae strain WHO was amplified by PCR and subcloned into the pET-30a vector to construct recombinant plasmid pET30a-PorB. Then the resultanting plasmid pET30a-PorB was transformed into E. coli BL21 （ DE3 ） and the expression of the Por B protein was in- duced with IPTG. The expressed PorB protein was purified by Ni 2 ＋ -chelating chromatography and de- tected by SDS-PAGE and Western blot. Results PorB gone was 1053 bp in length. SDS-PAGE anal- ysis indicated that the molecular weight of the recombinant protein was about 40 kD. The recombinant protein could react with polyclonal antibody against neisseria gonorrhoeae. Mouse antibodies against neisseria gonorrhoeae vaccine recognized rPorB, indicating that rPorB had a good immunogenicity. Conclusion The rPorB is successfully expressed and the indirect ELISA with it as coating antigen can be used to evaluate the titers of antibodies against neisseria gonorrhoeae vaccine. These results lay foundation for the diagnosis of neisseria gonorrhoeae infection and the research of vaccine of neisseria gonorrhoeae.