Cloning, Sequence Analysis And Construction Of Expression Vector Of Goat FSH Gene

The products of follicle stimulating hormone(FSH) used in animal husbandry are all extracted from the pituharies of bovine, goat, pig and some other animals. The lack of material and low purity of this kind of FSH limit its extensive application. By the means of gene engineering, large quantity and high purity FSH can be produced and the production expenditure also can be reduced, which is important to animal husbandry.Total RNA was extracted from pituitary of new butchered female goat for FSH-a and b subunit genes. cDNA were synthesized by RT-PCR reactions respectively and these cDNA were used as templates in PCR amplifications. The PCR products were 360bp and 390bp which just were the same length of the predicted goat FSH- a and P subunit genes. They were cloned to pMD 18-T respectively and selected two positive recombinants at random to analysis their sequences respectively, and compared their genes and amino acids with some other mammals. The results showed that the genes and amino acids of goat FSH- a and P subunits all have very high homology with other mammals and their homology all exceed 80%. These results proved that FSH- a subunit has high conservation among mammals and FSH- P subunit is also quite conservative among mammals although it shows species specificity. Goat FSH- a and P subunit genes were reamplified respectively by the new primers which had the corresponding restriction enzymes. The PCR products were purified and linked with pMD 18-T vector respectively. The positive recombinants were selected and digested with double restriction enzymes respectively. Goat FSH- a and P subunit genes which had sticky ends were purified and linked respectively with PF which also had the corresbonding sticky ends and were purified. The positive recombinants were selected finally. By this means, eukaryotic expression vectors of FSH single subunit-gFSH-a-PF, gFSH-b -PF and eukaryotic expression vectors of FSH double subunits-gFSH-a-b-PF, dFSH-a -gFSH- P -PF were all constructed successfully.In this experiment, gene cloning, sequence analysis and construction of expression vector of goat FSH were fist studied. This work initiated the basic research of recombinant goat FSH which supplied the theoretic basis and experiment references to its quantity production and application.Master: Zhang Li Major: Basic Veterinary Science Supervisor: Prof. Li Qing-zhang...