| SRY gene was the testis-determining gene of the male trait on the Y chromosome In mammals,and its function of sex differentiation in mammals is by the core HMG box a nd DNA combination.Therefore,the SRY sex-determining gene on the Y chromosome can be designed to produce a vaccine that produces an anti-Y sperm SRY vaccine to achieve sex control of mammalian.In this study,the target Sry was amplified by PCR from cow DNA samples extracted from the frozen semen of Simmental Cow with specified primers designed according to the sequences on database of Genbank.The nucleic acid vaccine w as prepared using vector pc DNA3.1-His-C-SRY.Ratio of the sexes in the offspring of fem ale and male rats was observed though injecting the antibodies into mice.The DNA of frozen spermatozoa of Simmental was extracted by two methods of the high salt method and the improved kit method.The detected concentrations of DNA wer e 151.16±25.09 ng/?L and 74.57±31.53 ng/?L,respectively.The PCR results of SRY gen e by high salt method was bright and the effect was good using the agarose gel electroph oresis(P<0.05).The Purified Products of SRY gene fragment was ligated to the p MD-19 T vector,after the bacterial solution PCR and Xho I,Bamh I double enzyme internal and e xternal treatment,and the correct recombinant plasmid p MD-19T-SRY was used as template,the SRY gene was amplified by primers with the cleavage site,and then the recombinant vector was constructed with the linear vector p ET28 a,the expression was identified after induction.The results show: The SRY gene sequence obtained by cloning and sequencing was 100% homologous to the SRY gene sequence published in Gene Bank.Construction of p ET28a-SRY expression vector by SDS-PAGE electrophoresis found that 30-33 KD betwee n the emergence of specific protein,but the negative control group p ET28 a did not appear in the protein,so to determine the specific protein for the SRY protein.PCR primers with restriction sites were designed according to the eukaryotic expressi on plasmid pc DNA3.1-His-C.After PCR amplification and double enzyme internal and ext ernal identification,and the correct recombinant plasmid pc DNA3.1-His-C-SRY was sequenc ed.The pc DNA3.1-His-C-SRY recombinant plasmid was extracted and transfected into He L a cells,after 48 h,the protein was cleaved and RNA was extracted.The expression of SR Y protein was detected by Western blotting and the SRY gene was amplified by RNA rev erse transcription.The results show:A specific band of about 27 k D was detected at the pr otein level and was consistent with the expected protein size of the SRY protein,demonstra ting that recombinant plasmids can be expressed in He La cells in mammals;At the level of RNA,SRY gene can be specifically amplified,which laid the foundation for the follow-up experiment.The recombinant eukaryotic recombinant plasmid pc DNA3.1-His-C-SRY was prepared in a large amount,and then the nucleic acid vaccine was prepared and the concentration was 1 ?g/?L.In vivo intramuscular injection of 100 ?g of recombinant plasmid passive i mmune maturation of two female mouses,once a week to strengthen the passive immune,a total of passive immunization 3 times.The control group was immunized with empty pl asmid and PBS in the same immunization mode and dose to the mature female mouse,th e number of each 10.After immunization,the eye was collected once a week,and the se rum was stored at-20 ?.The specific antibodies in the blood were detected by indirect ELISA.After immunization,mice were immunized with non-immunized male mice to calc ulate the male and female sex ratio of offspring.The results showed : Recombinant plasmi d pc DNA3.1-His-C-SRY produced an anti-SRY antibody after intramuscular injection of m ouse.Compared with the other groups,Ratio of the sexes in the offspring of female and male rats in the recombinant plasmid immunized group was 64:58(1.11: 1),so it was co ncluded that the sex of the newborn mouse might have shifted to the female. |
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