Genotypic Analysis, Construction of the Expression System and Immunological Identification of the Recombinant Proteins of the LipL32 Gene in the Dominant Serogroups of Leptospira interrogans in China

To investigate the existence of the major outer membrane protein (MOMP) gene LipL32 in 15 dominant Chinese strains of 15 serogroups of Leptospira interrogans and 2 international strains of 2 serogroups of Leptospira biflexa, and to clone and construct the expression system as well as to identify the recombinant proteins, genomic DNAs from strains of leptospira were prepared by routine phenol-chloroform method, and the fragments of the LipL32 gene with the whole length from the strains were amplified with high fidelity PCR. The target amplification products were sequenced after T-A cloning, and the expression system for the genes were thereby constructed. Expression of the recombinant proteins was identified by using SDS-PAGE after induction with IPTG at different dosages. Western blot assays with rabbit antiserum against the whole cell of TR/PatocⅠ of Leptospira and immunized serum with rMOMPs were used to determine the immunoreactivity and immunogenicity of the recombinant proteins. Microscopic agglutination test was used to determine the cross- agglutination titres in rabbit sera immunized with rMOMPs, and the cell adherence model of Leptospira was used to examine the blocking effects of rabbit antisera against these rMOMPs. It was found that the LipL32 gene could be found in all the 17 strains of Leptospira mentioned above with two different genotypes, i.e. LipL32/1 and LipL32/2. Amounts of expressions of rMOMP1 and rMOMP2 after IPTG accounted for 40% and 10% of the total bacterial proteins respectively. Both rMOMP1 and rMOMP2 could combine with the rabbit antiserum against leptospiral TR/PatocⅠ, and could induce the production of agglutination antibodies to these 17 strains of Leptospira with 1∶2 to 1∶64 MAT titres. The rabbit anti-rMOMP1 and anti-MOMP2 antibodies at 1∶2 to 1∶16 dilutions could efficiently block adherence of Leptospira. It concludes that all the Leptospira tested in the present study possess LipL32/1 or LipL32/2 genes, and the constructed expression system can express the rMOMP1 and rMOMP2. These recombinant proteins are showed to have good immunogenicity and satisfactory immunoreactivity.

赖型钩端螺旋体017株外膜蛋白LipL32基因重组质粒的构建及其细胞毒性的研究

目的构建赖型钩端螺旋体(钩体)017株外膜蛋白LipL32基因的重组质粒,并研究其细胞毒性。方法从钩体017株全基因组中用PCR方法扩增出目的基因,双酶切构建重组质粒,转化大肠杆菌,诱导表达LipL32蛋白,将表达的目的蛋白免疫新西兰大白兔,制备多克隆抗体,Western Blotting鉴定其免疫原性。将目的蛋白纯化、复性后作用于ECV304细胞,通过检测细胞的乳酸脱氢酶(LDH)、NO释放量研究其细胞毒性。结果扩增出816bp的LipL32基因,重组质粒经双酶切、PCR鉴定、测序均表明重组载体构建成功。经IPTG诱导表达的融合蛋白相对分子质量约52×103,主要以包涵体的形式表达,经免疫动物制备得到多克隆抗体,ELISA检测效价达1:32000,Western Blotting显示在目的蛋白位置处有特异性阳性条带。经过LipL32蛋白作用的ECV304细胞LDH、NO释放量和对照组比较有明显升高。结论成功构建LipL32基因重组质粒,该质粒能在大肠杆菌中表达,表达的目的蛋白对细胞有一定的毒性效应。

钩端螺旋体膜表面蛋白LipL41基因的克隆、序列分析及表达

目的：进一步分析钩端螺旋体LipL－41的免疫原性。方法：通过PCR的方法，以我国特有的钩端螺旋体流感伤寒群临海型lin6株（56609）的 DNA为模板，扩增目的基因 LipL41，克隆至 PcDNA3载体上，以自动测序仪测序后进行序列分析，然后克隆至原核表达载体pGEX－5T进行原核表达，Western印迹分析其免疫原性。珐票：获得长 1068 hp的片段，DNA序列分析表明该菌株的LipL41基因与文献报道具有很高的同源性（95．0％～99．4％），在大肠杆菌中获得了表达，并与抗钩端螺旋体血清反应。结论：该抗原为致病性钩端螺旋体所具有的保守性抗原成分，可能在钩端螺旋体病的诊断和预防中发挥作用。