| Vesicles are spherical or ellipsoidal particles formed by enclosing a volume of aqueous solution in a surfactant bilayer. Because of their structures similar to cell membrane, vesicles are used for membrane simulation, as well as the preparation of drug carrier. Follicle-stimulating hormone (FSH) plays an important part in the process of gametogenesis, differentiation, maturation and the appearance of behaviors related with the reproduction. The FSH preparation which can induce the ovulation of single oocyte and superovulation are extensively applied to the reproductive control and improving reproductive potency, but their source is limited and purity is low. In practice, recombinant follicle-stimulating hormone (rFSH) can avoid the deficiency of FSH prepared from pituitaries of domestic animals, and takes the chance to the extensive and efficient application. On basic research in goat rFSH in our laboratory, we transfered rFSH expression vector to Pichia pastoris via vesicles, in order to searching for the conducive ways of improving rFSH expression in vitro. In this study, the enhanced green fluorescent protein (EGFP) gene was constructed into the expression vector, which provided the necessary conditions for detecting transfection efficiency and expression of long-acting rFSH gene.According to the vesicles'characteristics and preparation methods, we used surfactant CTAB as the main material for preparation of vesicles. The yeast expression vector pPIC9K / FSHβ-CTP-αcontaining long-acting objective gene was digested by Sal I and CTAB can form vesicles in the presence of the linear vector. With the help of PI staining, we can observe vesicles'shape and that the vesicles parcelled vector into Pichia pastoris cells. The different concentrations of CTAB could have different toxic effect to yeast cells, so we need to determine the safe concentration of CTAB. After that, the ratio between CTAB and pPIC9K / FSHβ-CTP-αwas adjusted to prepare three groups of vesicular liquor, and use them to transfect Pichia pastoris competent cell GS115, with electric shock method for the control. The positive transformants His+ and multiple inserts were screened out by auxotrophic medium, antibiotic medium and PCR. Then the Mut+ phenotypes were screened out by contrasting Minimal Dextrose Medium (MD) and Minimal Methanol Medium (MM). His+ Mut+ phenotype transformants were inoculated Buffered Glycerol-complex Medium (BMGY) in conical flask to get enough Yeast cells. After achieving the required biomassto, replace the medium by Buffered Methanol-complex Medium (BMMY) including Methanol which concentration is 1.0% to induce the expression. Add pure methanol to a final concentration of 1.0% Methanol every 24 hours to maintain inducement and transfer 1mL of the expression culture to a 1.5mL microcentrifuge tube, which were used for the SDS-Polyacrylamide Gel Electrophoresis analysis. The expression level of FSHβ-CTP-αwas detected by Radio-immunoassay (RIA), thus, the difference between these two transfection methods can be obvious.In addition, according to the needs of the process of transfection, the reporter gene EGFP was inserted to the yeast expression vector pPIC9K / FSHβ-CTP-α. Using the plasmid pEGFP-N1 as the template to design a pair of primers with corresponding enzymatic sites of the vector pPIC9K but without stop codon of EGFP, special EGFP gene was amplified by PCR. Both of the amplified gene and vector pPIC9K / FSHβ-CTP-αwere double digested by SnaB I and EcoR I, then with the help of T4 DNA ligase, both of the reporter gene and objective gene were cloned into pPIC9K vector to obtain the yeast epression vector pPIC9K / EGFP-FSHβ-CTP-α. After identification by PCR and DNA sequencing, extract and linearize pPIC9K / EGFP-FSHβ-CTP-αand transfer it into GS115 by vesicle parceling method and electric shock method ibid. EGFP could be detected by fluorescence microscopy and flow cytometry(FCM).The result show that the yeast expression vector containing gene FSHβ-CTP-αcould induce the formation of vesicles from CTAB. Both of the reporter gene EGFP and objective gene FSHβ-CTP-αcould express protein effectively and respectively in Pichia pastoris through the method of vesicle parcelling transfection.We successfully constructed the yeast expression vector containing both the objective gene and reporter gene, and the experiment results approved that vesicle parcelling could be used in transfection studies as same as electric shock method. This study laid the the ground work for carrying out a further research on rFSH preparation and vesicle parceling in biological and medical applications.
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