Inhibin Gene Immunization Of Beef Cattle And Screening Of Differential Gene For Immune Response

In the practice of DNA immunization,it was found that nearly half of the large animals immunized with inhibin DNA vaccine did not respond to the vaccine and the specific reasons were not clear.In this study,we used the inhibin gene vaccine C501(pVAX-asd-IS)using attenuated Salmonella as a carrier to immunize Simmental cattle in order to investigate the effect of inhibin gene vaccine on reproduction,and screen differential genes associated with humoral immune responses,which could further improve the inhibin gene immunization technology and the effect of the vaccine,promote its promotion and application in the field of animal reproduction and lay the foundation for exploring the molecular mechanism of gene immune response.The main contents and results of the research are showed as follows: 1.Effect of inhibin gene immunization on follicular development and fetus in Simmental cattleA total of 41 Simmental cows were divided into two groups.Cows were injected with either 10 ml of PBS in the control group(n=11)or 10 ml of inhibin gene vaccine C501(pVAX-asd-IS)in the immunized group(n=30).Primary immunization was done on day 0.Boosting immunization was done on the 15 th day.On the 28 th day,the Ovysnch program was used for the simultaneous estrus-timed artificial insemination treatment.On the 29 th day,ultrasoundgraphy was performed once a day for 6 days and follicular development and ovulation were recorded.The results showed that: the antibody positive rate and antibody titer in the 4th week after immunization were significantly higher than that of the second week;the number of large follicles in the experimental group(2.46±0.22 vs 1.50±0.29,P <0.05)and diameter were higher than those of the control group(13.11±0.47 vs 11.92±0.86));the ovulation rate(92.9% vs 75%)and the fetus rate(85.71% vs 75%)of the cattles in natural estrus in the vaccine group were higher than those in the control group.2.Biosafety evaluation of inhibin gene vaccine on Simmental cattleAfter the immunization,the spirit and feeding situation of cows were observed.Cow dung,water and soil samples were collected on days 0,1,3,5,7,11,15,17,21,35 after immunization,respectively.InvA,crp,asd,and INH gene were detected by PCR.It was found that there was no abnormality in the cattle after immunization and InvA,crp,asd,and INH gene were not detected in cow dung,water,and soil samples.The results showed that after intramuscular injection of the INH DNA vaccine,the vaccine would not spread to the outside through the cattle and would not adversely affect the environment.3.Study on differential genes of humoral immune response in Simmental cattle induced by inhibin gene immunizationAccording to the level of antibodies,the blood samples were collected from 3 cows for each of the control,high and low immune response group.Differentially expressed genes were analyzed by RNA-seq.The results showed that there were 97 differentially expressed genes between the high and the low-response immunization group,198 differentially expressed genes between the control and the low response group and 127 differentially expressed genes between the high response group and the control group.Among them,IFITM1,BOLA-DQA5,BOLA-DQB,IL11 RA and TSLP were key genes that caused differences in immune response.