[Soluble expression, purification and immunoreactive identification of mouse zona pellucida 3 fusion protein].
Mammalian zona pellucida 3(ZP3) plays an important role in the induction of capacitating sperm acrosome reaction. In this study, we obtained the soluble mZP3 fusion protein and identified its immunoreactivity. mZP3 cDNA was cloned into plasmid pMAL-p2x, and the recombinant plasmid was transformed into Escherichia coli BL21. To get the soluble mZP3 fusion protein, we tried to optimize the expression conditions, including additives, IPTG concentrations, temperatures and induction duration. Then, Western blotting and ELISA were used to identify the immunoreactivity of the purified protein. Based on the optimization experiments, we concluded that the best soluble expression conditions for the mZP3 fusion protein involved incubation to an A600 of 0.6, addition of glucose to a final concentration of 0.02 mol/L, addition of IPTG to a final concentration of 0.6 mmol/L and then further incubation for 4 h at 25 degrees C. Western blotting and ELISA showed that the mZP3 fusion protein retained immunoreactivity. The fusion protein can be used as solubility antigens for developing the immunocontraception vaccines of mZP3 and detecting the immune effects of the vaccine.
Sun
Meiyu
M
Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumchi 830046, China.
Ma
Zhenghai
Z
Li
Yongxin
Y
Lü
Tao
T
Chen
Kaixu
K
Zhang
Fuchun
F
D000818:Animals; D004527:Egg Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D051379:Mice; D011956:Receptors, Cell Surface / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D012995:Solubility; D022642:Vaccines, Contraceptive / Q000276:immunology; D000072216:Zona Pellucida Glycoproteins
D004527:Egg Proteins; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D022642:Vaccines, Contraceptive; D000072216:Zona Pellucida Glycoproteins; C000606521:Zp3 protein, mouse