Cloning, Sequence Analysis And Prokaryotic Expression Of Lactate Dehydrogenase C4(LDH-C4) From Chipmunk(Tamias Sibiricus)

The chipmunk(Tamias sibiricus) is a species of rodents in the Squirrel family. They are mainly distributed in the many areas of the Northeast China, North China, northern Shaanxi and Qinling region which is used to live in the woodland, farmland, or terraced areas. The fruit and seeds are their mainly food, and some chipmunks will eat plant seedings and a little insect. Chipmunks have the habits of storing food and often forget storage locations, resulting waste. Since they reproduce fast, distribute widely, and have a big and wide appetite, chipmunks have bring serious damage to China’s forestry. Usually, the traditional prevention method is totally depending on artificial capture and poison bait which is time-consuming and inefficient. Now the immune infertility technology is becoming the key factor to control the amount of rodent pests. At its core, we can control the key factor of sperm-egg recognition or binding. So it’s crucial for us to obtain the efficient auto antigen. Scientists have improved the excellent performance about LDH-C4 as an auto antigen. In this study, chipmunk LDH-C4 was selected as the research object. Through molecular biology techniques the coding sequence of LDH-C was cloned and LDH-C4 protein was expressed in E. coli. This study provides a theoretical basis for future immune-contraceptive study on chipmunk control.Based on preliminary experiment, specific primers were designed according to the LDH-C mRNA sequence of spermophilus tridecemlineatus in GenBank. Using total RNA isolated from chipmunk(Tamias sibiricus) testis, the cDNA encoding chipmunk LDH-C was amplified by RT-PCR for the first time and was submitted to GenBank(Accession number:KJ513014). The chipmunk LDH-C cDNA contains an open reading frame of 999 nucleotides and encodes a polypeptide of 332 amino acid residues. Nucleotide identity between Tamias sibiricus and spermophilus tridecemlineatus was 97%. The molecular weight was predicted to be 37 kD with an isoelectric point at 7.04. Phylogenetic tree analysis suggested Tamias sibiricus had the closest phylogenetic relationship with the spermophilus tridecemlineatus, followed by other rodents. Second structure prediction revealed the presence of 40.96% alpha helices, 24.40% random coils, 24.10% extended strand and 10.54% beta turn. Three-dimensional structure of LDH-C4 was predicted by homology modeling methods after compared with proteins of known structure.According to chipmunk LDH-C cDNA sequence, specific primers with restriction sites were designed to amplify LDH-C. Restriction enzyme digestion and sequencing verified that prokaryotic expression vector pET-28a-LDH-C was successfully constructed by homologous recombination technique. Prokaryotic expression vector pET-28a-LDH-C was transformed into E. coli BL21(DE3). Expression of protein was then induced by addition of IPTG. SDS-PAGE and Western-blot revealed that we successfully expressed the chipmunk LDH-C4.