Rapid Cloning, Expression And The Preliminary Study On Directed Evolution In Vitro Of Luffin-b Gene From The Seeds Of Luffa Cylindrical

Objective: To obtain bioactive recombinant luffin-b protein by rapidly cloning luffin-b gene from the seeds of Luffa cylindrical and the expression vector pET-28a (+) in E.coli, and to establish mutant Lib of luffin-b gene by E.P PCR.Methods:(1) To design and synthesis a pair of primers according to the cDNA sequence of luffin-b gene alreadly reported by Kataoka in 1992. And to extract the genome of Luffa cylindrical.The DNA sequence encoding luffin-b was cloned from the fresh seeds of Luffa cylindrical by genome PCR. The target DNA fragments were sequenced after T-A cloning to identify whether luffin-b gene has any introns or not.(2) The luffin-b cloning plasmid was constructed by inserting the luffin-b gene into vector pUCm-T.(3) The luffin-b expression plasmid was constructed by inserting the luffin-b gene into vector pET-28a (+).At 16℃,30℃and 37℃, luffin-b was expressed in E.coli by 1.0 mmol/L IPTG induction. After12-16h, the recombinant luffin-b was identified by SDS-PAGE.(4) The DNA sequence encoding luffin-b was cloned from the fresh seeds of Luffa cylindrical by E.P PCR.Results:(1) In this work, the coding sequence of the gene was shown to be identical with that determined by Kataoka. In comparison with the reported luffin-b, the homology of nucleotide sequence of the cloned luffin-b gene was 100%.It was identified that luffin-b gene has no intron.(2) The luffin-b cloning plasmid was successfully constructed by inserting the luffin-b gene into vector pUCm-T.(3) The luffin-b expression plasmid was successfully constructed by inserting the luffin-b gene into vector pET-28a (+). The production of recombinant protein was analyzed by SDS-PAGE, which indated that the product at 16℃was more than others.(4) The mutant Lib was primarily constructed.Conclusions: The identification that there is no intron in luffin-b gene by genome PCR provides a convenient and feasible way for the further research. Moreover, the mutation of luffin-b gene by EP PCR also provides experimental basis and establishes a preliminary platform on the directed evolution in vitro of luffin-b protein.