Expression and preliminary use of human sperm-specific lactate dehydrogenase in Escherichia coli

Abstract

Objective To construct a recombinant vector of sperm-specific human lactate dehydrogenase ( hLDH-C4 ), express it in Escherichia coli ( E. coli ) BL21 ( DE3 ) and utilize it in the detection of anti-sperm antibody. Methods The coding sequence of hLDH-C4 was amplified from human testis λTripIEx cDNA library, and inserted into pET-28a( + ) after restriction enzyme digestion with Hind Ⅲ and Xho Ⅰ. The resultant recombinant vector was used to transform E. coli BL21 ( DE3 ) and the His-Tag fused hLDH-C4 was expressed after induction with IPTG. Western blot was used to analyzed the recombinant protein and LDH activity of bacterial lysates was determined. An indirect ELISA method for the detection of anti-sperm antibody was established by using the recombinant hLDH-C4 as antigen matrix. Results pET-28a( + )-hLDHC was successfully established. The protein with size of 35kD could be induced by IPTG when the recombinant plasmid was transfected into E. coli BL21 ( DE3 ). Western blot showed that the recombinant protein could be specifically recognized beth by anti-His tag monoclonal antibody and by rabbit anti-human LDH-C4 antibody. In addition, the recombinant protein showed high-level LDH activity when the bacterial lysate after IPIG induction was used to check LDH activity. The recombinant hLDH-C4 was confirmed when it was used in indirect EL1SA to detect anti-hLDH-C4 antibody. Conclusions The coding sequence of hLDH-C4 is cloned into the vector pET-28a( + ) and recombinant hLDH-C4 was expressed at a high level in E. coli. The recombinant hLDH-C4 is useful in the detection of anti-sperm antibody. Key words: L-lactate dehydrogenase; Isoenzyme; Spermatozoa; Autoantibodies; Escherichia coli; Recombinant proteins