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.
ABSTRACT
Objective To investigate molecular diagnostic method for hereditarymethemoblobinemia. Methods The cDNA coding sequence of NADH-cytochrome b5 reductase (b5R) from 3 patients with hereditary methemoglobinemia was analyzed by direct sequencing of RT-PCR products and the genomic DNA of b5R gene by PCR-restriction endonuclease digestion or PCR-sequencing. Results The b5R cDNA of patient A was T/C heterozygous at nucleotide 527 and G/A heterozygous at nucleotide 608. The b5R cDNA of patient B was G/A heterozygous at both nucleotide 170 and nucleotide 179. The b5R cDNA of patient C was G/A heterozygous at nucleotide 608 and C/T heterozygous at nucleotide 791. Result of genomic DNA analysis was in agreement with that of cDNA approach. Conclusion The method for molecular diagnosis of hereditary methemoglobinemia was established and 3 novel b5R gene mutations were identified in compound heterozygosity in 3 Chinese patients.
ABSTRACT
Objective To express Japanese encephalitis virus (JEV) E protein in Escherichia coli. Methods The gene coding for JEV E protein was amplified by using RT-PCR, and cloned into plasmid pET-28a. The constructed plasmid was transformed into E.coli BL21 (DE3). Expression of E protein by the transformants was induced by IPTG and analyzed by SDS-PAGE and Western blot. Results Five nucleotide changes leading to amino substitutions were identify in the E protein gene of our laboratory strain of JEV when compared to a published gene sequence of JEV E protein. The yield of expressed JEV E protein with relative molecular mass approximately 52?10 3 was 35% of total bacterial proteins. Conclusions JEV E protein was expressed successfully in E.coli, which should be useful for the production of diagnostic reagents and the analysis of gene structure/function of the E protein.