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[Construction of prokaryotic expression vector of hALR and its expression in E.Coli]
1Institute for Viral Hepatitis, Chongqing University of Medical Science. Chongqing 400010, China.
Objective:
To construct prokaryotic expression vector of hALR and express rhALR in E.Coli. through PCR and recombinant gene technology.
Methods:
The coding region with Nde I and BamH I sites of hALR was obtained from pUC19-hALR constructed with PCR method. PCR product and plasmid pET11a were digested by corresponding endonucleases, respectively. The fragments cut were ligated by T(4) DNA ligase to gain recombinant expression vector. The recombinant plasmid pET11a-hALR was electrotransformed into BL(21) (DE(3)) strain. The rhALR was expressed in the bacteria under induction of IPTG.
Results:
Endonucleases digesting and DNA sequening confirmed that the coding region of hALR was correctly inserted into the vector. The rhALR was successfully expressed in E.Coli. Its MW. With 1. 5A10(4) was in correspondence with theoretic value and its amount is 30% of total bacteria protein. It existed not only in supernatant but also in precipitation of broken bacteria.
Conclusion:
The successes in construction of expressive vector of hALR and in expression of rhALR in E.Coli. make it possible to study further on its biological functions and antibody preparation.
