Cloning And Expression Of The Linked Fragment Of C1 And C3 Constant Regions Of Envelope Gene From Human Immunodeficiency Virus 1 SF2 Strain In E.coli DH_5α

Objective: to construct the clone of the linked fragment of Cl and C3 constant regions of envelope gene from human immunodeficiency virus 1 SF2strain and express it in E.coli DHsa so as to facilitate a further study of the structure and function of the fusiou protein. Methods: the recombinant plasmid 9BiR6 containing the HIV-1 proviral genome of SF2 strain was chosen as the template. Two fragments of Cl and C3 constant region of envelope gene were amplified by PCR with several primer pairs designed with the aid of DNAstar Softeware, respectively,The two amplified fragments digested by Kpn I were then ligated by T4 ligase. Furthermore the linked fragment modified by EcoR I and Sail was inserted into the multiple cloning site (MCS) of glutathione S-transferase(GST) of plasmid pGEX-4T-2 . The recombinant plasmid (named pGEX-4T-2/C IC3) was identified by restriction endonuclease assay and its sequence was determined by dideoxy-chain termination method. Finally ,the recombinant plasmid pGEX-4T-2/ C1C3 wastransformed by TSS method into E.coli DH5a and induced with Isopropyl-B -D-thioglactoside (IPIG) to express GST-C1C3 fusion protein init . Then the expressed proteins in E.coli DHsa were analyzed with SDS-PAGE electrophoresis.Results: the recombinant pGEX-4T-2/C1C3 was obtained. Its sequence analysis showed that the reading frame of it is correct and no mismatched baseses happened. And the fusion protein of GST-C1C3 was efficiently expressed in E.coli DH5a of which relative molecular weight (Mr) is about 43 X 103 Daltons.Conclusion : therefore the success of construction and expression of the recombinant plasmid pGEX-4T-2/ C1C3 has indicated that not only could the expression of fusion protein in E.coliDH5a rollout a basis for the study of its activity, but also the above work has laid a neccessary foundation for future research on new vaccine against HIV-1...