The antigenic determinants of Vp7 gene of porcine rotavirus A were amplified from cells infected with rotavirus by the reverse transcription-polymerase chain reaction (RT-PCR), and a 981bp cDNA segment was acquived. U...The antigenic determinants of Vp7 gene of porcine rotavirus A were amplified from cells infected with rotavirus by the reverse transcription-polymerase chain reaction (RT-PCR), and a 981bp cDNA segment was acquived. Using T-A cloning technique, the PCR product was cloned into pGEM-T vector. Cloning plasmid pGEM-T-Vp7 and the prokaryotic expression shuttle vector between E.coli and Lactobacillus pW425t were digested by SacI and KpnI double enzymes, respectively. The purified Vp7 gene was subcloned into the expression vector pW425t. Thus,the recombinant pW425t-Vp7 was constructed, and 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 37.07 ku exogenous protein was observed and it was about 15% of the total protein . 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 Vp7 gene in prevention of porcine rotavirus.展开更多
文摘The antigenic determinants of Vp7 gene of porcine rotavirus A were amplified from cells infected with rotavirus by the reverse transcription-polymerase chain reaction (RT-PCR), and a 981bp cDNA segment was acquived. Using T-A cloning technique, the PCR product was cloned into pGEM-T vector. Cloning plasmid pGEM-T-Vp7 and the prokaryotic expression shuttle vector between E.coli and Lactobacillus pW425t were digested by SacI and KpnI double enzymes, respectively. The purified Vp7 gene was subcloned into the expression vector pW425t. Thus,the recombinant pW425t-Vp7 was constructed, and 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 37.07 ku exogenous protein was observed and it was about 15% of the total protein . 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 Vp7 gene in prevention of porcine rotavirus.
