The Application Of Soe Pcr In The Lovastatin Nonaketide Synthase Gene Cloning

The gene splicing by overlap extension PCR, shorter form SOE PCR, which is a technique that introduce the primers with complementary terminus to overlap the products of PCR, then the different fragments can be spliced by extending the overlapping chains. Lovastatin, also know as monacolin K, which is one of inhibitors for HMG-COA reductase comes from microorganism and the drug for high cholesterol, so it can reduce the incidence of arteriosclerosis, myocardial infarction, coronary disease, etc., therefore, it is significant in medicine. In order to verify the application of SOE PCR in the lovastatin nonaketide synthase gene (LovB) cloning,4 couples of primers were designed to amplify 4 exons of LovB, after that the exons can be connected by overlapping PCR one by one to form the cDNA sequence of LovB①-④, which could be linked with T vector after purified, then the recombinant was transformed into DH5a. Lastly the positive transformant was sieved out by PCR and indentified by sequencing further. The results indicated that the exons of LovB①-④with the size of 1300bp, was amplified successfully by overlapping PCR, and was linked with T vector after purified. The recombinant was transformed into DH5a and screened on the LB medium with Amp roughly, after that the positive recombinant was identified by PCR. Then the sequencing result was blasted with the LovB cDNA sequence titled AF151722.1 in the NCBI, it showed the 98.5% homoeology, as the result of the LovB①-④-TA recombinant plasmid was constructed successfully, which laid a solid foundation on expressing the LovB gene in Pichia pastoris futher.