Studies On Expression Of Recombinant ApoCⅢ In Pichia Pastoris

Apoprotein-CⅢ(ApoCⅢ) has held the attention of molecular biology scientists as it plays an important role in metabolism of blood lipids.The human apoCⅢgene is located in a gene cluster together with the apoAⅠand apoAⅣgenes on the long arm of chromosome 11 and is about 3.1 kb . The human apoCⅢgene is expressed mostly in the liver and partly in intestine. It is controlled by positive and negative regulatory elements that are spread throughout the gene cluster. The human apoCⅢgene has 4 exons while it has 3 introns. IntronⅠis 625bp, intronⅡis 135bp, and intronⅢis 1837bp. They are all very close to the introns of the other apoCs.The cDNA of apoCⅢhas been isolated and sequenced, is 519bp. Furthermore, the length of RNA of apoCⅢis about 532bp to 538bp, including 49bp nontranslation frame in 5-terminal, 297bp protein coding region, terminal codon TGA and about 183-189bp which is nontranslation frame, with a poly-A tail.In this study, we dedicate in these domains:①Cloning of apoCⅢgene with RT-PCR and construction of cloning plasmid pMD18-T-apoCⅢ;②Construction of expression plasmid pPICZα-apoCⅢand transformed it into Pichia Pastoris via electroporation. Afte induced with methanol, we obtained r-ApoCⅢin the culture supernatant of the Pichia pastoris.1. Cloning of apoCⅢDNA sequenceWe firstly obtained the DNA sequence encoding apoCⅢby RT-PCR, and the template RNA derives from the human liver. Then the purified PCR production was joint to pMD18-T vector. The results of sequencing showed that the recombinant cloning vector pMD18-T- apoCⅢwas constructed correctly.2. Construction of recombinant ApoCⅢexpression plasmid(1) Construction of ApoCⅢexpression vector pPICZα-apoCⅢWith the recombinant plasmid of pMD18-T-apoCⅢas template, the sequence encoding template was obtained by PCR. The endonuclease sites of XhoI and EcoRI were added into two ends of apoCⅢsequence. Jointing the sequence to pPICZαvector after being digested with these two endonucleases. The results of sequencing indicated that the construction was correct.(2) Transformation pPICZα-apoC3 into Pichia pastoris We transformed the purified pPICZα-apoCⅢvector into Pichia pastoris and then it cultured on YPD plates containing 100μg/ml Zeocin. After 48 hours, dozens of colonies of transformants formed.3. Expression of recombinant ApoCⅢin Pichia pastoris Selected several clones from YPD/Zeocin plates, and induced them with 0.5% methanol in BMMY media. Analyzed the expression product on by SDS-PAGE.As mentioned above,the expression plasmid pPICZα-apoCⅢand Pichia Pastoris high efficient expression system for rhApoCⅢwas constructed successfully. And it established a significant foundation for the deep researching.