Application Of Proteomic Methods For Identification Of ASA Cervical Mucus In Dairy Cows

Cows immune infertility seriously affect the cows normal reproduction, and it is moredifficult to diagnosis than other types of infertility. It is very important that the quantitativeand qualitative analysis for the difference of ASA between fertility and infertility cows. In thisstudy, We conduct a study of the cervical mucus of infertile and pregnant cows bySDS-PAGE,display Western blot and two-dimensional electrophoresis analysis,and constructedan effective indirect ELISA method.The results are as follows:1. A kind of effective method of separation of protein in dairy cows’s cervical mucus bySDS-PAGE was established, optimizing conditions were8%separation glue,15μL sample,100V, electrophoresis for3-4h. A protein, which the molecular weight is22-25KD,preliminarily determined is ASA Light Chain,was gotten. Infertility cows contained ASALight Chain in cervical mucus were100%（24/24）, fertility cows contain ASA Light Chainin cervical mucus were14.2%（2/14）, but the expression level of ASA Light Chain was lowerthan that of infertility cows.2. Western blot test results show that cervical mucus ASA of infertile cows, themolecular weight88-90KD,66KD,55-57KD,25-27KD sperm membrane antigen havespecific immune response, antigen recognition rate was100%(24/24),100%(24/24),87.5%(21/24),70.8%(17/24) respectively; fertile cow cervical mucus ASA molecular weight88-90KD,55-57KD,25-27KD identification was only21.4%(3/14),14.2%(2/14),14.2%(2/14), fertile cow cervical mucus does not recognize66KD sperm membrane antigen.3.The optimal conditions of indirect ELISA were determined. The reactions wereoptimized, including5μg/mL coating antigen of sperm membrane protein,1:5dilution oftesting cervical mucus, the optimal incubation time of cervical mucus and enzyme-labeledsecond antibody were1h and1.5h respectively. OD490nmvalues higher than0.513weredetermined as ASA positive immune infertility standard for the ELISA and lower than0.410as fertility standard, OD490nmvalue was not more than0.410but less than0.513asdoubtful.The assay was highly specific and showed no no cross-reaction with the positivecervical mucus of other bovine diseases. The variant coefficient of optical density value in abatch and between batches varied from4.39%to9.31%, and from4.08%to9.96%,respectively. Compared with TAT testing, the results indicated that the coincidence, sensitivityand specificity of indirect ELISA was97.8%,100%and97.6%, respectively. 4. Infertile dairy cervical mucus protein on the sample size for0.8,1.6mg,0.8mg ofprotein, less sample volume, the electrophoresis pattern small individual protein spots, anddoes not clear;1.6mg protein electrophoresis pattern of each protein point is a large, thick.When can the cervical mucus of fertile and sterile cows protein sample were1.6mg canpregnant cows and infertile cows cervical mucus protein two-dimensional electrophoresismaps were given protein spots67and151infertile cow cervical mucus protein spotssignificantly more than pregnant cows.