目前，多数外源基因的克隆与表达，主要采用大肠杆菌。然而，由于大肠杆菌能够引起人类和动物发生不同程度的腹泻，导致机体损伤，所以其表达产物需要经过复杂的分离纯化才能达到食品医药标准。与之相比，乳酸菌作为人和动物肠道内正常...目前，多数外源基因的克隆与表达，主要采用大肠杆菌。然而，由于大肠杆菌能够引起人类和动物发生不同程度的腹泻，导致机体损伤，所以其表达产物需要经过复杂的分离纯化才能达到食品医药标准。与之相比，乳酸菌作为人和动物肠道内正常菌群之一，已被证明具有诸多益生功能，而且被公认为安全无毒的（Generally regarded as safe，GRAS）。展开更多
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
文摘目前，多数外源基因的克隆与表达，主要采用大肠杆菌。然而，由于大肠杆菌能够引起人类和动物发生不同程度的腹泻，导致机体损伤，所以其表达产物需要经过复杂的分离纯化才能达到食品医药标准。与之相比，乳酸菌作为人和动物肠道内正常菌群之一，已被证明具有诸多益生功能，而且被公认为安全无毒的（Generally regarded as safe，GRAS）。
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.
