钩端螺旋体外膜脂蛋白LipL32基因的克隆和表达及其在ELISA检测中的应用

Clonging and Expression of Leptospiral Outer Membrane Protein LipL32 Gene and Application of Recombinant Antigen in Enzyme-linked Immunosorbent Assays

Objective\ To construct L32 pQE32 recombinant expression vectors, and to induce the expression of recombinant Leptospiral outer membrane protein LipL32. Establish method of recombinant Leptospiral outer membrane protein based ELISA. Method\ Gene coding of Leptospiral LipL32 protein was amplified by PCR,then recombinant cloning vectors pGEM T/L32 and expression vectors L32 pQE32 were constructed. Recombinant expression vector was transformed into the competent host E.coli. DH 5α and E.coli. M15. Recombinant Leptospiral LipL32 protein was expressed by IPTG induced method. Immulon microtiter plates were coated at 37℃ overnight with 100 ng of purified recombinant protein per well, 3 positive and 4 negative sera were used in indirect ELISA. Results\ Mature Leptospiral LipL32 gene fragment about 750 bp was amplified by PCR. LipL32 gene was inserted into expression vectors pQE32, the molecular weight of fusion protein was corresponding to the estimated molecular size of mature Leptospiral LipL32 protein. Results of Western blot and ELISA demonstrated intense LipL32 reactivity with anti Leptospira sera. Conclusion\ findings indicate that recombinant Leptospiral LipL32 may be an important, useful antigen for the serodiagnosis of Leptospira.