Cloning, Expression And Biological Characteristics Study Of Human Sperm FSCB

The process of fertilization in mammalian consists of a series of complicated events. The spermatozoa must have a nomal ability of motion which helps the gametes meet each other and sperm penetrate the egg. Before mammalian spermatozoa gain the fertilization capability, they must undergo the physiological and morphologic changes in female genital tract, named capacitation. Capacitation is an important physiological event before acrosome reaction and fertilization.There are many proteins related with capacitation, such as SP-10, FA-1, YLP-12, CABYR, etc. The protein FSCB(Fibrous Sheath CABYR-Binding,FSCB) found by Dr.Li YF in 2007 located in the fibrous sheath of mouse sperm tail and could bind with CABYR protein. Now FSCB have been demonstrated be an important protein involved in the regulation of sperm capacitation. Based on study of mouse sperm FSCB, the bioinformatics of human sperm protein FSCB were analyzed. The results suggested that complete sequence information of human FSCB had been submitted on GenBank and it located on chromosome No.14.So far, there is no any report about human FSCB. Because the mouse FSCB could play an important role in sperm motion and capacitation, it would be significant to carry out the research on biological characteristics and functions of human FSCB.The human full-length and truncated FSCB(FSCBt) gene were amplificated by PCR, then the recombinant expression vectors were constructed by inserting them into plasmids pET32a(+) and pET28b(+). After the recombinant proteins of FSCB and FSCBt were dissociated and purified, the polyclonal antibody against human sperm FSCB were prepared successfully by immunizing rabbits. Then the preliminary research on its biological characteristics was conducted. The main results and conclusions are as follows:1. Cloning and sequencing of human FSCB gene: Based on theoretical sequence of FSCB gene, primers with situs of appropriate restricted incision enzyme were designed, then the full-length and its truncated sequence(FSCBt) were abtained by PCR amplification. The sequencing results are coincident with GenBank data.2. Construction of human FSCB expression vectors and their prokaryotic expression: Firstly, FSCB and FSCBt genes were inserted respectively into expression vector pET32a(+) and pET28b(+), subsequently the recombinant plasmids were transformed into the E.coli BL21. Finally, the two recombinant strains were constructed and the expression of the recombinant proteins was induced by IPTG successfully. Their apparent molecule masses were about 230 kDa and 30 kDa respectively, which were significant greater than their theoretical values.3. Dissociation and purification of the recombinant protein FSCBt and preparation of the polyclonal antibody against FSCB: Because of the existence of His-tag in the expression plasmids, the fusion protein FSCBt could be purified by Ni2+ chromatographic column. After additional sieve chromatography, highly purifed recombinant protein FSCBt was abtained with a purity coefficient of 95%. According to the standard protocol of inoculating animal, the mixture of purified FSCBt and Freund's adjuvant was injected subcutaneous into the female rabbits. The polyclonal antibody was abtained. The specificity of this PAb recognizing the recombinant proteins FSCB and FSCBt was confirmed by Western blot.4. Tissue specificity assay of expression of human FSCB: The expression of FSCB in multiple tissue including cardiac muscle, lung, pancreas, spleen, muscle and testis was observed and its specific expression in testis was confirmed using the PAb by Western blot.5. Expression of FSCB in human testis: The expression of FSCB in spermatozoa and some spermatids of human testis tissue were demonstrated using the PAb by immunohistochemistry.6. The preliminary study on the localization of human FSCB: The specific localization study on sperm using the adult ejaculated semen showed the human FSCB was confined in the principle piece of sperm tail by indirect immunofluorescence analysis.In summary, the recombinant human FSCB was expressed by prokaryotic expression systems and purified by multiple purification systems, and the specific polyclonal antibody was prepared successfully. The specific expression and localization of FSCB on human sperm was observed. These preliminary results laid a good foundation for further research on its biologic functions in sperm motion and capacitation, and would be helpful to understand the complicated molecular mechanism of capacitation.