[Expression of recombinant Vp6 gene of porcine rotavirus A with non-antibiotic Lactobacillus vector in Escherichia coli].
The antigenic determinants of Vp6 gene of porcine rotavirus A was amplified from infected MA 104 cell by the reverse transcription-polymerase chain reaction (RT-PCR), the product of which was a 1194bp cDNA segment. Using T-A cloning technique, the PCR product was cloned into pGEM-T vector. Cloning plasmid pGEM-T-Vp6 and the prokaryotic shuttle expression vector pW425t between E. coli and Lactobacillus, were digested by SacI and KpnI double enzymes, respectively. The purified Vp6 gene was subcloned into the expression vector pW425t. Thus, the recombinant pW425t-Vp6 was constructed, which then was transformed into the competence thyA gene-mutant E. coli X13. Treated lysates of bacterium were loaded directly onto SDS-PAGE, on which approximately 44.88 kD fusion protein was observed. The protein was further analyzed using Western blot, which indicated that the protein was reactive with the antibody of rotavirus A. The results lay foundation for further studies on the Lactobacillus subunit vaccine and DNA vaccine of Vp6 gene for prevention and control of porcine rotavirus.
Wang
Chun-feng
CF
College of Animal Science and Technology, Jilin Agricultural University, Changchun, China. wchunfeng@hotmail.com
Cong
Yan-long
YL
Liu
Shang-gao
SG
Li
Yu
Y
D000818:Animals; D000956:Antigens, Viral / Q000235:genetics*; D036022:Capsid Proteins / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D007778:Lactobacillus / Q000235:genetics*; D010957:Plasmids; D011995:Recombination, Genetic; D012401:Rotavirus / Q000235:genetics*; D022243:Rotavirus Vaccines / Q000276:immunology; D013552:Swine; D019444:Vaccines, DNA / Q000276:immunology
D000956:Antigens, Viral; D036022:Capsid Proteins; D022243:Rotavirus Vaccines; C061332:VP6 protein, Rotavirus; D019444:Vaccines, DNA