| Zona pellucida (ZP), a complex matrix surrounding oocytes, is comprised of three immunologically distinct glycoproteins (ZP1, ZP2, ZP3). Zona pellucida 3 (ZP3) is a major glycoprotein of the zona pellucida that possesses the sperm receptor function and the acrosome-inducing activity. It induces antibody that can block sperm/oocyte interaction. So ZP3 has long been used as a candidate antigen to develop an immunocontraceptive vaccine. One of the yeast -- Pichia pastoris has been developed to be an outstanding host for the production of foreign proteins. It offers many advantages, for example, it does not have the endotoxin problem associated with bacteria nor the viral contamination problem of proteins produced in animal cell culture. Furthermore, P. pastoris expression system has strong promoter that provides high expresstion. The proteins produced in P. pastoris are typically folded correctly and secreted into the medium. A number of proteins have been produced using this system, such as tetanus toxin fragment, human serum albumin and lysozyme. This study mainly aimed at the expression of Lagurus lagurus zona pellucida3 (lZP3) in Pichia pastoris system, and its purification and characterization. Firstly, a pair of primers were designed according to the lZP3 gene constructed in our lab (GenBank NumberAF515621). The core fragment of lZP3 was amplified by PCR. Then the target gene and the vector pSuperY were linked together at two restrictive sites: EcoRI and XbalI. After kanamycin screening and mini-preparation, the positive clones were testified with the same restrictive enzymes. In addition, the plasmid was sequenced and blasted in GenBank comfirming the correctness of the plasmid used as an expression vector. Then the expression plasmid was transformed into SMD1168 by electroporation after linearized with AvrII. Zeocin was used to screen positive clones that inserted into yeast genome. A band about 55kD was observed on SDS-PAGE, and then Western blot analysis was conducted furtherly to confirm that the band was specific lZP3 glycoprotein. This expressed protein was then used to immunize animal to produce antiserum. Antiserum against the ZP3 was determined with ELISA and Western blot analysis, and the titer was above 1:50000. In order to test immunological activity of the Lagurus lagurus zona pellucida3 (lZP3) expressed in Pichia pastoris, female NIH mice were immunized with lZP3. The NIH mice were chosen and enlisted to 4 groups randomly, and were received immunization with three kinds of vaccine: pCMV4-lZP3 DNA vaccine, lZP3 protein vaccine, pCMV4-lZP3 DNA and lZP3 protein co-immunization. Each injection, per mouse received 100ug/100ul of pCMV4-lZP3 at hind leg or 100ug/100ul lZP3 protein with Freund's adjuvant under skin. Sera were collected from the postorbital vein after each immunization. lZP3 antiserum levels were detected by ELISA. 5 weeks later, nonimmunized male mice were introduced to immunized females and allowed to mate with them. The result was that the levels of lZP3 anti-serum in mice were rising with the increasing of the immunization times. 83.3%, 87.5%, 100% infertility rate was showed in DNA, protein and co-immunization, respectively, while it was 0% in non-immunized group. There was a significant difference between the number of pups per litter in the immunization group and in the control group (p(0.01). These results encourage us to further ZP3 immunocontraceptive function study and ZP3 vaccine development.In this study, isoelectric point of Lagurus lagurus zona pellucida3 (lZP3) protein expressed in Pichia pastoris was also assayed by Mini-IEF with Model 111 Mini-IEF Cell. Preliminarily purifying of lZP3 was performed with sulfate and dialysis, and 80% sulfate was suitable for separating lZP3 protein. After dialysis in 20mM Tris-HCL overnight to eliminate sulfate, the purified protein was added to the IEF gel to analysis its isoelectric point. A band between pI 6.0 and 7.0 was seen in the gel. This result was consistent with predicted pI (6.34) of lZP3. Put these...
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