Cloning Of Trichosanthin And Its Mutant Expression In E.coli

Objective: Trichosanthin (TCS) is a kind of Ribosome-inactivating protein(RIP). Itmainly functions in inhibiting protein synthesis. TCS has broad-spectrum activity againstvirus and, in recent study, it can kill the cells infected by HIV, but not the normal ones.Successfully, We have amplified the gene of TCS through Trichosanthes kiritounfMaxim genomic DNA by PCR and obtained TCS recombinant clones and expressionplasmid. We have established TCS random mutant library, and therefore, it facilitates theprokaryotic expression of TCS.Methods: 1). TCS gene was amplified from Trichosanthes kiritounf Maxim genomicDNA by PCR, and then it was cloned into pUCm-T. After sequencing analysis, theacquired recombinant plasmid was transformed into E.coli DH5a;2). The CDS of TCS was amplified by PCR and transferred into pET-28a(+) afterdigested by Nco I and Hind III; the acquired recombinant plasmid was transformed intoE.coli DH5a; The target protein was expressed by inducing with IPTG in BL21(DE3).Then, the protein was identified by SDS-PAGE.3). Establish TCS and its activity centre mutant library; Induce protein expression withIPTG and use SDS-PAGE for analysis.Results: We successfully cloned TCS gene and its recombinant plasmids. There isobvious protein band after prokaryotic expression. Random mutant library of TCS hasbeen established.Conclusion: The establishment of the TCS mutant random library and TCS recombinantplasmids expressed in BL21(DE3) could be a solid foundation for further researching andscreening medicines against HIV by gene engineering.