Study On Cloning And Expression Of Merozoite Surface Antigen 2 Locus(MSA-2c) Of Babesia Bovis Isolated In Xinjiang And Antigenicity Of Its Recombinant Protein

The babesiosis is a tick-borne zoonosis blood protozoiasis. The bovine babesiosis is reportedly the one of the severest parasitosis that does harm to the world cattle industry. The impressionable neutralization antibody was induced to generate by bovine babesiosis merozoite surface antigen 2c (MSA-2c) protein with strong immunogenicity and conservative B-cell phaenotype (Wilkowsky, et al., 2003). This is believed that MSA-2c protein can be used to be a stable diagnosis instrument for bovine babesiosis detection, as well as a candidate material for producing bovine babesiosis genetic engineering subunit vaccine. Therefore, many studies on prevention of bovine babesiosis have been focused on MSA-2c gene and its coding protein.The following three studies were conducted in the present article.1 MAS-2c gene cloning and sequence analysisThe specificity primer was designed through bovine babesiosis gene sequence released in GenBank. Xinjiang bovine babesiosis MSA-2c gene was obtained by PCR with whole blood gene template from a sick cattle. The results show that Xinjiang bovine babesiosis MSA-2c gene is an open reading frame with 798bp, according on which its molecular weight was deduced to be 29kDa with 267 amino-acid residues. It was discovered that there is one base mutation in Xinjiang bovine babesiosis compared with the international standard strain gene, and no change was found in protein sequence through homological analysis of DNA sequence and protein sequence. It is first that the candidate gene MSA-2c for bovine babesiosis vaccine was successfully cloned in China.2 Prokaryotic expression, purification of recombinant protein and its immunoreactivity of bovine babesiosis MSA-2cThe recombinant prokaryotic vector pGEX-4T2/MSA-2c was constructed, and multicopy recombinant strain was obtained by transforming pGEX-4T2/MSA-2c to E coli with CaCl2. Multicopy recombinant strain induced to express with IPTG was purified by glutathione sepharose 4B, then SDS-PAGE and Western-Blot was respectively conducted to show that the induced pGEX-4T2/MSA-2c positive strain is immunoreactivitive recombinant protein with an expression of 55ku. The protein antibody potency of subject mouse by immune of recombinant was found to be more than 1:200,000 through ELISA, which strongly supports that MSA-2c protein can be used to be a stable diagnosis instrument for bovine babesiosis detection, as well as a candidate material for producing bovine babesiosis genetic engineering subunit vaccine.3 Eucaryotic transient expression, purification of recombinant plasmid and its immunoreactivity of bovine babesiosis MSA-2cThe recombinant plasmid pCDNA3.1+/MSA-2c was injected into the subject mouse vena caudalis, the eukaryotic expression was conducted in vivo, and the total RNA was extracted from liver, the transient expression of the recombinant plasmid pCDNA3.1+/MSA-2c was detected through RT-PCR. After four times intramuscular injection, the target strip was obtained through Western-Blot with the positive blood serum as antibody, and the purified GST fusion protein as antigen. The antibody potency of subject mouse by immune of recombinant plsmid was found to be more than 1:100,000 through ELISA, which strongly supports that the recombinant plasmid pCDNA3.1+/MSA-2c is immunoreactivitive as DNA vaccine.