Expression of Core Domain of Porcine Zona Pellucida 3β in E.coli

To obtain the recombinant core domain of porcine zone pellucida 3β （cZP3β） for the further research on its functions Methods The nucleotide sequence region from 44 to 306 codons of pZP3β entire eDNA was obtained by PCR and then was cloned into pET-3c vector. After being identified, recon was transformed into E.coli BL21 （DE3） pLysS and then induced by IPTG. Results The recombinant cZP3β was expressed in E. coli up to 15% of total cellular proteins, and was made sure by Western blot analysis. Conclusion The research on expression of core domain of pZP3β could benefit to further investigation of its immunogenicity and the development of antigen preparation.

Expression of Nonfusion Extracellular Porcine Zona Pellucida Protein 3β in E.coli

Objeetive To obtain the recombinant non fusion extracellular porcine zona pellucida protein 3β （pZP3β） in E.coli Methods By modificated the transition initiation region （TIR） in primers, synthetic nucleotide was gained by PCR. Such gene was cloned into pET-3c vector and transformed into E.coli BL21（DE3）pLysS. Results The recombi.ant nonfusion extracelhtlar pZP3β was expressed in E.coli to 10% of total cellular proteins, and identified by the Western blot method. Conclusion Modification of nucleotide without changing amino acid sequences is an effective means to increase non fusion expression rate of recombinant proteins, such as pZP3β in E. coll.

Therapeutic Effect of Bushen Huoxue Recipe(补肾活血方) on Autoimmune Premature Ovarian Failure Mice Established by Immunization with Recombinant Porcine Zona Pellucida 4 Antigen

Objective： To investigate the efficacy and mechanism of Bushen Huoxue Recipe （补肾活血方, BHR） in the treatment of mudne autoimmune premature ovarian failure （POF）. Methods： The recombinant porcine zona pellucida 4 （pZP4） was expressed in E. coli BL21 （DE3） strain within prokaryotic plasmid pET28a （＋）, purified by Ni-affinity chromatography and verified by Western blot. Murine autoimmune POF model was established by immunization with pZP4 of female BALB/c mice. Fifty POF mice were randomly divided into 5 groups, which were respectively given low （3.75 mg/kg）, moderate （7.5 mg/kg）, and high dose （15.0 mg/kg） of BHR by gastrogavage once daily for 20 days, with 17-13-estradiol （0.13 mg/kg） and normal saline as positive and negative control. Estrous cycles were analyzed through vaginal smears, serum estradiol （E2） levels, and anti-pZP4 antibody titers were detected by ELISA. The proliferative responses in vitro of spleen lymphocytes to pZP4 antigen restimulation were measured by [3H]-thymidine incorporation, and the histomorphology changes of ovary were evaluated by optical microscope. ]Results： The purified pZP4 was visible as a single lane with 14.4 kD in SDS-PAGE and Western blot. The murine POF model with lengthening estrous cycles, decreased levels of serum E2, high titers of serum anti-pZP4 antibody, and reduced ovarian follicles and corpus lutea were established by immunization with recombinant pZP4. Treatment with moderate and high dosage BHR significantly increased ovarian follicles and reduced the proliferation of spleen lymphocytes to the pZP4 antigen of POF mice （P〈0.05）. However, only the high dosage BHR administration significantly improved the estrous cycles, elevated the serum E2 levels （P〈0.01）, and decreased the serum anti-pZP4 antibody titers of model mice （P〈0.05）. Conclusions： The recombinant pZP4 could evoke the antigen-specific immune response in mice and induce the autoimmune ovarian injury. It has been demonstrated that BHR was able to increase the serum E2 levels and protect ovarian functions from the autoimmune injury in murine POF model.