ABSTRACT
In the present study, the fusion gene lipL32/1-lipL41/2 from Leptospira interrogans was constructed by using PCR with linking primers, expressed in prokaryotic expression system. And the immuno-reactivity of its expressed product was determined. Meanwhile, SDS-PAGE and BioRad agarose image analyzer system was used to examine the expression output of the target recombinant protein rLipL32/1-LipL41/2, and the immune-reactivity of this recombinant protein was identified by Western blot assay. ELISA was used to detect the level of antibodies against the recombinant proteins in sera of patients with leptospirosis. It was demonstrated that the percentages of similarity in nucleotide and the putative amino acid sequences of the fusion gene lipL32/1-lipL41/2 with the corresponding sequences previously reported were 99. 9% and 98. 9%respectively. The expression output of the target recombinant protein was approximately 10% of the total bacterial proteins.Both the rabbit antisera against rLipL32/1 and rLip41/2 could recognize and combine to the recombinant fusion protein rLip32/1-Lip41/2 as well. The positive rates of antibodies in 228 leptospirosis patients with the recombinant proteins rLip32/1,rLip41/2 and rLip32/1-Lip41.2 were 97.4%, 78. 5% and 99. 1% respectively. The results of the present study leads to the conclusion that the fusion gene lipL32/1-lipL41/3 with its prokaryotic expression system is successively constructed and the expressed recombinant protein shows excellent immuno-reactivity, thus suggesting that it may be used as the antigenic candidate for the development of the leptospiral genus-specific engineering vaccine and for the preparation of diagnostic kits.
ABSTRACT
Objective To construct single chain antibody specific to membrane protein of Schistosoma japonicum by gonetic engineering technique. Methods The V\-H (heavy-chain variable region) and V\-L (light-chain variable region) genes were amplified by PCR from the genomic DNA of NP11-4 cell line, and sequenced by Sanger's method. The ScFv was constructed in pTHA90 vector using V\-H and V\-L genes, then expressed by IPTG. Results The V\-H and V\-L genes were obtained through PCR. The DNA sequences showed that V\-H and V\-L were new variable region genes of antibody. They were registered by GenBank. A ScFv gene with (Gly4Ser) 3 intralinker in the pTHA90 vector was successfully constructed. The ScFv was expressed as thioredoxin-fused proteins about 36^2 kDa. Conclusion A specific ScFv against the membrane protein of Schistosoma japonicum was constructed and expressed.