Cloning Of Flanking Region Of Pig PSP-Ⅱ Gene And Construction Of Accessory Sex Gland Specific Expression Vector

Among all of the boar spermadhesin proteins, PSP-â…¡is a major component of boar seminal plasma, which is a specific secretary product of accessory sex gland. In order to construct accessory sex gland specific expression vectors, we cloned the sequences of 5'- and 3'-flanking region of PSP-â…¡by PCR technology, according to the published sequence of PSP-â…¡, and the length of cloned sequences is about 4.1kb and 3.1kb. The two cloned fragments were inserted into Pmd18-T vector and sequenced by Shanghai GeneCore BioTechnologies Co., Ltd. subsequently. The sequences were analysis by Blast process, the result is that the homology was 99.8%, 100% of frist 500bp of the 5'- and 3'-flanking region of PSP-â…¡, indicated that the sequence we cloned was correctly.In order to construct accessory sex gland bioreactor, the 5'-and 3'-flanking region of PSP-â…¡were cloned and inserted into modified pcDNA3.0 and formed the accessory sex gland specific expression vector of EGFP gene. To test the expression character of the accessory sex gland specific expression vector, the recombinat vector of PC-EGFP and the control pEGFP-N1 vector were transfected with the mouse seminal vesicle directly by injection. 48 hours after injection, mouse seminal vesicle was sampled and the total mRNA was extracted to test EGFP gene expressiong by RT-PCR and observed fluorescin under fluorescence microscope by made frozen section. The resualt showed that pC-EGFP vector was expressed. In order to detect pC-EGFP vector's expression characteristic in detail, pC-EGFP vector parceled with PEI was injected into ICR mouse by tail vein, after 48 hours seminal vesicle, liver, heart, lung and muscle tissue were sampled, extract the total mRNA to test EGFP gene expressiong by RT-PCR and frozen section was made, and then the fluorescence was observed under fluorescence microscope immediatedly. The result showed that the vector was seminal vesicle specific. This paper indicate that seminal vesicle specific vector was constructed succefully. This construction established the base for deeper research of pig accessory sex gland specific expression system.