Study On Cloning Of AQP7 Gene And Preparation Of Polyclonal Antibody In Dairy Goat

Dairy goat industry is one of the important members of China’s dairy industry.Goat milk is favored by consumers,but its basic ewe quantity and quality are seriously insufficient,which directly affects the development of the industry.Sex control technology has been widely used in many animals because it can effectively control the gender of newborn animals.However,at present,there is a lack of theoretical evidence,it is less used in the dairy goat industry,and the effect is not good.It needs in-depth research to solve the existing scientific problems.The results obtained will be directly applied to dairy goats and promote the development of dairy goat industry.The previous study of the research group found that there was a significant difference in aquaporin 7(AQP7)between X sperm and Y sperm of dairy goats.In this study,the full-length CDS region of AQP7 gene was cloned from the testis of dairy goats.The prokaryotic expression vector was successfully constructed,and the recombinant protein was expressed and purified.The purified recombinant protein was used to immunize New Zealand white rabbits to obtain polyclonal antibody,and the expression of AQP7 protein in X and Y sperm was detected,which laid a foundation for the study of AQP7 protein and the control of sperm gender through AQP7.The results are as follows:1.The 1077 bp sequence of AQP7 gene of dairy goat was cloned,and the total length of CDS region of the gene was 993 bp,encoding 330 amino acids.Bioinformatics analysis of the CDS region of the gene showed that the C-terminal secondary structure of the protein was rich;TMHMM predicted that the protein had three extracellular regions;Finally,combined with the prediction results of open reading frame(ORF),the 339 bp gene sequence suitable for antigen fragment was obtained,and the gene sequence of the antigen fragment was cloned.Homology analysis showed that the sequence had low homology with rabbit and could be used as immune peptide.The proportion of rare codons in the peptide was analyzed,and BL21(DE3)competent cells suitable for expressing recombinant protein were selected.2.p ET32a(+)-AQP7 recombinant vector was constructed.5900 bp vector sequence band and 339 bp antigen fragment gene sequence band were identified by double enzyme digestion.The 30 k D AQP7 recombinant protein was induced and expressed.The recombinant protein was induced by IPTG with the optimum expression condition of 0.5mmol/l for 6 h.The protein expression form was explored.It was found that the recombinant protein was expressed in the form of inclusion body in the precipitation.The inclusion body was broken and the protein was denatured with 8 M urea,The recombinant protein was affinity purified with Ni NTA resin.The target band of SDS-PAGE showed that the purification effect was good.The purified protein was renatured by gradient dialysis.After renaturation,the mass concentration of AQP7 recombinant protein was determined by BCA method ≥ 2.0 mg / ml.3.The polyclonal antibody of milk goat AQP7 was prepared,and the antibody titer of1:128000 was detected by I-ELISA.Western blot showed that the antibody still had good specific recognition of antigen protein when diluted 5000 times.The antibody could recognize AQP7 protein in natural testicular tissue of dairy goat,and the protein differential expression of AQP7 gene was detected in X sperm and Y sperm protein.The content of AQP7 protein in Y sperm was high;Immunofluorescence on milk goat sperm showed that the antibody could bind to sperm,and the difference of fluorescence intensity was observed in X sperm and Y sperm,indicating that AQP7 protein was located in sperm acrosome;Then the testicular tissue sections of dairy goats were made and immunofluorescence staining showed that the expression of the protein changed dynamically in each stage of spermatogenesis,and the expression was the highest after differentiation into long sperm.To sum up,this study successfully cloned the dairy goat AQP7 gene,purified the AQP7 recombinant protein,and prepared the dairy goat AQP7 polyclonal antibody.Using this antibody,it was detected that the expression of AQP7 gene was different on the X and Y sperm of dairy goat,which provided theoretical basis and molecular tools for the follow-up study of dairy goat AQP7 gene.