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The Preliminary Study on Construction, Expression and Immune Response of the Recombinant Human CatSper1 Specific Antigen

Author: HuangDan
Tutor: PanShanPei
School: Jinan University
Course: Developmental Biology
Keywords: Sperm CatSper1 CatSper1 specific antigen Prokaryotic expression Immunocontraception
CLC: R392.1
Type: Master's thesis
Year: 2010
Downloads: 42
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Abstract


Aim:To construct the prokaryotic expression vectors of recombinant human Catsperl-specific antigen (including the extracellular region, the pore region and the promiscuous T cell epitope TT580-599) and express the recombinant antigens. To evaluate antibody response, antifertility effect of immunization with the recombinant hCatsperl-specific antigen and accumulate data for further study of immunocontraception targetting hCatsperl.Methods:The DNA fragment of human Catsperl coding the transmembrane region (S1-S6) was amplified by RT-PCR with total mRNA extracted from human testis.The DNA fragment of human Catsperl-specific antigen was synthesed by overlapping PCR and inserted into prokaryotic expression vector pET-21b and pET-21b-Trx, constructing the recombinant expression plasmids pET-21 b-hCatsperl and pET-21 b-Trx-hCatsperl. After identified by restriction endonuclease digestion, PCR and sequence analysis, the recombinant plasmids were transformed into E. Coli BL21 (DE3) and the recombinant proteins were expressed with IPTG induction. The expressed proteins were analysed by Tricine-SDS-PAGE and Western blotting. The inclusion bodies of hCatSperl specific antigen were separated by Tricine-SDS-PAGE. After staining, destaining and equilibrating with phosphate buffer, the gel containing hCatsperl-specific antigen were cut, rubbed, emulsified with freund’s adjuvant and used to immune male rabbits by multi-point subdermal injection and muscle injection. The recombinant Trx-hCatsperl specific antigen was separated and purified from inclusion body by denatured with 2 mol/L urea and Ni-chelating chromatography. Anti-hCatSperl specific antigen antibody response of the immunized rabbits were monitored by ELISA with the purified Trx-Catsperl specific antigen as coating antigen. Reactions of antiserum specific to recombinant Trx-hCatsperl and human testis tissue protein were detected by Western blotting. Antiserum’s binding to human sperm were detected by indirect immunofluorescence (ⅡF). The effect of antiserum on human sperm motility was determined by co-culturing sperm with antiserum in vitro, and recording the sperm moving trajectory using continuous exposure photography. Paraffin sections of the rabbits testis and epididymis were prepared and stained by HE to observe pathological changes in testis and epididymis of immunized rabbits.Results:The prokaryotic plasmid pET-21b-hCatsperl specific antigen and pET-21b-Trx-hCatsperlspecific antigen were successfully constructed. Results of PCR, restriction endonuclease digestion and sequence analysis showed that the length and sequence of inserted DNA fragment coincided with those of the designed recombinant hCatsperl specific antigen. The recombinant proteins were expressed after the recombinant plasmid transformed to E. coli BL21(DE3). Analysis by Tricine-SDS-PAGE and Western blotting showed that recombinant human CatSperl specific antigen and the recombinant Trx-hCatsperl specific antigen were mainly in inclusion bodies, with 11kDa、24kDa size respectively and consistent with the expected protein size. The pure soluble recombinant Trx-hCatsperl specific antigen was separated and purified by denaturation, Ni-chelating chromatography and renaturation from inclusion body. Antibody response could be induced in male rabbit by immunization with recombinant hCatSperl-specific antigen. By Western blotting, antiserum was able to bind to Trx-hCatsperl specific antigen and also bind to human testis protein showing a band with size-80kDa consistent with the size of CatSperl protein. Antiserum could bind to the human sperm, showing green fluorescence in the middle, tail and the region behind the head. Antiserum could depress human sperm motility in a certain extent in vitro.No pathological changes were found in the testis and epididymis of immunized rabbits. Spermatogenic cells in defferent developing stages and with normal appearance could be observed in testicular seminiferous tubules. The epididymis tubules exhibited integrated histological structureand were full of sperm.Conclusion:The prokaryotic plasmid pET-21b-hCatsperlspecific antigen and pET-21b-Trx-hCatsperlspecific antigen were constructed and recombinant proteins were successfully expressed in prokaryotic system. Anti-hCatSperl specific antigens antibody response was induced after immunization of New Zealand male rabbits with the recombinant hCatsperl-specific antigen. The antiserum was able to react with the 80kDa protein in human testis and could bind to human sperm. The antiserum treatment in vitro could depress sperm motility in a certain extent. No significant testis and epididymis pathological injury can be seen in the immunizesd rabbits during this study. The antifertility effect of immunization with the recombinant hCatSperl-specific antigen needs to be further studied.

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