Objectives    To construct the recombinant plasmid pPIC9K FSH-Occludin for preparing a multivalent contraceptive vaccine

which interfering the function of Sertoli cells in the mouse testis.     Methods    After isolated the total RNA from mouse brain and testis tissues

the specific region of follicle stimulating hormone-b (FSH-b) subunit and the secondary extracellular domain of occludin were amplified by RT-PCR

respectively. The catenation fragments of FSH-occludin were obtained through double PCR amplification and cloned into the expression vector PPIC9K of P. pastoris to construct 2 different recombin

ant plasmids (pPIC9K FSH-occludin). The recombinant vector was introduced into P. pastoris strain GS115 by electroporation and selected with G418 for FSH-occludin gene integration 1. Super G418 resistant clones were selected and screened for FSH-occludin gene expression. The FSH-occludin gene was successfully cloned into pPIC9K.     Results    The recombinant plasmids (pPIC9K FSH-occludin) were identified by DNA sequencing and double enzymatic digestion with EcoRⅠ and NotⅠ. It was confirmed that the reconstruct plasmids containing the same sequences as the theoretical designing. The results of  SDS-PAGE showed that the molecular weight of the expressed product was 1.4×10

3

and 1.2×10

3

.    Conclusion    The results indicate successful construction of recombinant plasmids containing specific regions of FSH and occludin. It can be used for further study on the immune contraceptive effect in vivo.