To obtain the protein of duck interferon alpha and study its biological activities, the prokaryotic expression vector of DuIFN-αwas constructed and expressed in BL21 (DE3) plysS. Using PCR technique, the protein gene...To obtain the protein of duck interferon alpha and study its biological activities, the prokaryotic expression vector of DuIFN-αwas constructed and expressed in BL21 (DE3) plysS. Using PCR technique, the protein gene of DuIFN-αwas cloned from pMD-18-duIFN-αrecombinant. The gene was then inserted to pGEM-T vector and identified by restriction endonuclease analysis and sequencing. DuIFN-αwas ligated with the prokaryotic expression vector of pET30 a, then transformed into BL21 (DE3) plysS. The best inducing time and IPTG concentration for the expression of this recombinant protein was tested through the expression of the positive recombinant with different time span and different IPTG concentration. Lots of the protein of DuIFN-αwere expressed in BL21(DE3)plysS with 1 mmol·L-1 IPTG for 4 hours and its molecular weight for 34 000.展开更多
[Objective] The paper was to improve the preparation efficacy of Taq DNA polymerase. [Method] Ni column was used to purify Taq DNA polymerase carrying with 6xHis tag,and recombined vector. Using the thermal-resistant ...[Objective] The paper was to improve the preparation efficacy of Taq DNA polymerase. [Method] Ni column was used to purify Taq DNA polymerase carrying with 6xHis tag,and recombined vector. Using the thermal-resistant characteristics of Taq DNA polymerase,the crude extract was treated at 75 ℃ for 1 h,and the activity of prepared enzyme solution was verified by PCR test. [Result] The recombinant pET-32A-Taq could highly express in BL21 （DE3） host bacteria and remove hybrid protein by thermal denaturation. The enzyme preparation with the activity further higher than purchased Taq DNA polymerase was obtained. [Conclusion] Taq DNA polymerase prepared by thermal purification method is simple with low cost,and can meet the needs of a large number of conventional PCR amplification.展开更多
基金Studying Abroad and Coming Back Home Fund (LC02C08).
文摘To obtain the protein of duck interferon alpha and study its biological activities, the prokaryotic expression vector of DuIFN-αwas constructed and expressed in BL21 (DE3) plysS. Using PCR technique, the protein gene of DuIFN-αwas cloned from pMD-18-duIFN-αrecombinant. The gene was then inserted to pGEM-T vector and identified by restriction endonuclease analysis and sequencing. DuIFN-αwas ligated with the prokaryotic expression vector of pET30 a, then transformed into BL21 (DE3) plysS. The best inducing time and IPTG concentration for the expression of this recombinant protein was tested through the expression of the positive recombinant with different time span and different IPTG concentration. Lots of the protein of DuIFN-αwere expressed in BL21(DE3)plysS with 1 mmol·L-1 IPTG for 4 hours and its molecular weight for 34 000.
基金Supported by National Natural Science Foundation of China (30872254)~~
文摘[Objective] The paper was to improve the preparation efficacy of Taq DNA polymerase. [Method] Ni column was used to purify Taq DNA polymerase carrying with 6xHis tag,and recombined vector. Using the thermal-resistant characteristics of Taq DNA polymerase,the crude extract was treated at 75 ℃ for 1 h,and the activity of prepared enzyme solution was verified by PCR test. [Result] The recombinant pET-32A-Taq could highly express in BL21 （DE3） host bacteria and remove hybrid protein by thermal denaturation. The enzyme preparation with the activity further higher than purchased Taq DNA polymerase was obtained. [Conclusion] Taq DNA polymerase prepared by thermal purification method is simple with low cost,and can meet the needs of a large number of conventional PCR amplification.
