Cloning And Expression Analysis Of Rabbit Follicle-Stimulating Hormone β(FSH β) Gene

Follicle Stimulating Hormone is a glycoprotein hormone synthesized and secreted by animals pituitary basophilic cells which composed of alpha and beta subunits. The beta subunit is determinants of Follicle Stimulating Hormone biological activity and immunological activity. There are some studies have shown that FSH beta gene polymorphism was closely associated with animal characters of litter size, and that is one of the major genes affect the animals give birth. In this study, as the object to New Zealand white rabbits, cloning, prokaryotic expression, real-time PCR and other methods used in to study FSHβ gene expression in a prokaryotic expression system, preparation of polyclonal antibody anti-rabbit FSHβ, and FSHβ genes differentially expressed in rabbit different reproductive organs such as ovaries, fallopian tubes, uterus, testis and vas deferens. The main results are as follows:1. Designed a pair of primers of New Zealand white rabbits FSHβ gene refer to the FSHβ gene mRNA sequence of Japanese rabbits in GenBank database, cloning and analysis the sequence, then compared to other species’ CDS sequences and amino acid sequence. The results showed that, the CDS sequence cloned exactly same with the sequence of FSH beta gene from GenBank database, the homology of the nucleotide sequence with other mammalian is betwwen86%to90%, but representing low with poultry and fish,74.1%and53.3%, respectively. Cluster analysis based on the the FSHβ gene, we can see that rabbits has closer relationship with human, chimpanzees; these results are basically same with biological classification results. 2. PCR amplification FSHβ gene without signal peptide sequence, and successfully constructed a prokaryotic expression vector called pGEX-4T-1-FSH β. We abtain fusion proteins which molecular weight42kDa approximately, after some operate as follows:induced expression with IPTG, broken the bacterial body by ultrasonic wave, SDS-PAGE gel electrophoresis. The product expressed by the recombinant plasmid in E. coli BL21(DE3) mainly in the form of inclusion bodies. Immunize mice after purified the inclusion body protein and separate the serum to get the polyclonal antibody. ELISA and Western Blot test results showed that the titer of antibody is1:256000and well of specificity.3. Real-time quantitative PCR analysis the expression levels of FSHβ gene in reproductive organs such as the9to23-week-old rabbit ovaries, fallopian tubes, uterus, testis and vas deferens. The results showed that the FSH beta gene can be expressed in five reproductive organs, but the difference was not significant (P>0.05) in different tissues. There are some differences in different weeks of age. Substantially, in13to15weeks of age higher expression were significantly different (P<0.05) than other weeks of age. The difference was not significant (P>0.05) among other weeks of age. Therefore, the FSH beta gene can be expressed in reproductive tissues and the highest expression level in the puberty.