The Gene Engineering Expression Of Carbonic Anhydrase 4 And Effect Of The Expression Product On Chicken Sperm Motility

Utero-vaginal junction and infundibulum existed special tubular glands, which could make sperm reside for a long time in hen repreductive tracts and keep fertilization for long periods.This tubular glands were called sperm storage tubules(SSTs). Meanwhile,we found CA Ⅳ play an important role in regulating animal sperm maturation and storage. Thus, in this experiment, CAⅣ was chosen to disscuss the impact of sperm motility in SST.According to the sequence of chicken CA Ⅳ gene, the real-time PCR was established to detect the CAIV expression in chicken reproductive tract, including infundibulum, magnum, isthmus, uterus, utero-vagina junction and vagina. The results showed that CAⅣ expression was the highest in the utero-vaginal junction, and its expression was higher than that of the infundibulum(p<0.05). CAⅣ expression of the utero-vaginal junction and infundibulum was higher than that of magnum, isthmus, vagina and uterus,respectively(p<0.05). High expression of CAⅣ in utero-vaginal junction and infundibulum suggested that CAⅣ may play an important role in avian sperm storage.According to the m RNA sequence of chicken CAⅣ and codon bias of Pichia pastoris, the modified DNA sequence for chicken CAⅣ were synthesized, the sequence were cloned into p PICZαA vector to construct the recombinant,and then the recombinant plasmids were transfected into Pichia pastoris GS115 by electroporation. The positive recombinant strains were inducted to express protein by addition of 1 % methanol, and purified by Ni ion affinity chromatography. SDS-polyaerylamide gel electrophoresis(SDS –PAGE)and Western Blot of His-tag protein analysis were used to detect the fusion protein. The results showed that recombinant expression vector p PICZαA-CAⅣ was constructed and chicken CAⅣ protein(approximately 36 k Da) was expressed successfully, and high purity target protein was obtained through Ni ion affinity chromatography.After adding the purified CAⅣ protein into chicken fresh semen, 2 h、6 h、12 h and 24 h later, p H of semen and sperm motilily were detected, and physiological salineno was as control group. Results showed that the p H value and sperm motility of the two groups were decreased significantly(p<0.05). At 6h, 12 h and 24 h after seman storing, the p H value of CAⅣ protein group was significantly lower than the control group(p<0.05). At 12 h and 24 h after seman storing, the sperm motility of CAⅣ protein group was significantly lower than the control group(p<0.05). These results indicated CAⅣ protein may inhibit sperm motility by regulation of the p H value.