Clonging Of LfcinB And Lactoferrin N-lobe Gene And The Study On Expression Of N-lobe Gene In Pichia Pastoris

Lactoferrin is an iron-binding glycoprotein of transferrin family and found in milk, tear, saliva and other secretions. It was proved that this protein has multifunctional properties in vitro and may be involved in regulation of iron homeostasis, inhibition of bacterical and virious growth, immunilogical regulation and activation of genetic transcription through previous studies. It was pay more attention. So, sciencists had found that lactoferrin from cow milk display greatest bioactivity than other mammalian's lactoferrin. In addition, many functions of lactoferrin from various mammalian were thought to be involved in the N-lobe of their protein,especially Lactoferricin(Lfcin) from N-lobe.Pichia Pastoris is a newly developed yeast expression system in recent years. It combines the growth characteristics and molecular genetic background of prokaryotic organisms together with post-translational modifications of eukaryotes, such as: glycosylation and disulfide bond formation et al. More than 300 different proteins have been successfully expressed in the Pichia Pastoris expression system during the past 20 years.Object: To set up an effective expression system in vitro by genetic engineering in order to know bovine lactoferricin(Lfcin B)and bovine lactoferrin N-lobe biological activities well,find out their applications and establish the foundation for later.Methods: This experiment synthesized two pairs primer according to the reported sequence of bovine lactoferrin mRNA in GenBank (NO:NM₁80998), and extracted the total RAN from lactating bovine mammary grand. The cDNA encoding bovine lactoferricin(Lfcin B)and bovine lactoferrin N-lobe was cloned by RT-PCR method. The obtained DNA fragment was then cloned into pGM-T vector, after transforming into E. coli DH5α, its plasmid DNA was prepared and tested by restriction enzymes and sequencing and correct results was named pGM-LfcinB and pGM-N-lobe. Positive recombinant plasmid——pGM-N-lobe was digested with EcoRI and XbaI and the N-lobe fragment was recovered,at the same time, the Pichia pastoris expression Vector pPICZαA was also digested with EcoRI and XbaI, then this two digested DNA fragment was ligated by T4 DNA ligase and tested by restriction enzymes and sequencing. It was confirmed that the foreign gene had been inserted into the expression plasmid and the ORF was right. The expression pasmid was electroporated into Pichia Pastoris GS115 after digested by Sac I .The positive transformants was selected on YPDS plates at 30℃for several days, and decided the phenotypes of methanol utilization of transformants by comparing the growth conditions on MM and MD plates. Genomic DNA of recombinants was isolated from digested yeast cells by lyticase and was used to further confirmation if the hLF gene had been integrated into the yeast genome by PCR. According to the phenotypes of His+ transformants, the positive clones were cultured in BMGY/BMMY, inducing by methanol. We collected the supernatants and cell pellets by centrifugation, precipitated the protein by TCA, and detected the expression of foreign protein by SDS-PAGE,anatibacterial assayResults: The sequencing results of recombinant clone vector are as follows:the length of LfcinB cDNA and bovine N-lobe cDNA is respectively 87bp and 1028bp,and comparison with sequences registered in GenBank(NO:NM₁80998) shows 100% homology in LfcinB cDNA sequence and more than 99% homology in bovine N-lobe cDNA sequence; The remcombinants was induced for target protein expressing and detection of foreign protein indicated bovine N-lobe gene was not expressed in Pichia pastoris GS115 strain.Conclusion: we successfully cloned bovine LfcinB and bovine lactoferrin N-lobe base sequence and constructed expression plasmid pPICZ—N-lobe。Although ours interest protein failed to express, we explored the conditions of yeast growth, transformation, screening, induction and detecting of foreign protein. As a result, which provides bases for later experiment.