Study On Pregnancy Diagnosis Technology Of Dairy Cows Based On Pregnancy-associated Glycoprotein PAG1

Early and accurate diagnosis of cow's pregnancy is a key issue in dairy farming.The implantation of the cow's embryo was initiated from 19 to 20 days after artificial insemination(AI)and the process was completed approximately 42 days.After the implantation of the embryo and contact with the maternal body,substances that can enter the maternal body are generated,which is detected from the maternal body fluid by passing through the system to various body fluids of the body.It is possible to test certain molecules to diagnose pregnancy.So 20 days 42 days after AI is the most suitable time period for early diagnosis of cows.Many molecules play an important role in the process of implantation.These molecules are also potential markers of pregnancy diagnosis.There are periodic changes in Pregnancy Associated Glycoproteins(PAGs)in the serum of Artiodactyla,despite Function of PAGs is not yet clear,but a lot of studies have been done where as markers for early pregnancy diagnosis.In this study,pregnancy-associated glycoprotein1(PAG1)was selected as marker molecule to establish a ELISA kit.Firstly,according to the amino acid sequence of PAG1,sequence of GTPPQEFQVC was selected to synthesize polypeptide,which was coupled with KLH for animal immunity,and screened out monoclonal cells by coupling with BSA,and name 2017-020-KLH and2017-020-BSA.Four female BALB/c mice were immunized,and the titers of the mice with the highest titers were selected(the titre was up to 12800),and the blood samples were collected from the orbit to determine the titers.Then the spleen cells of the mice and the mouse osteocytoma SP2/0 were fused by method of PEG.The fusion cells were screened and cultured in semi-solid medium(including HAT),and the positive monoclonal cells were screened twice by ELISA,and four positive monoclonal cell lines #1,#3,#7,#12 were selected.The subtypes of these monoclonal cells were identified.The results showed that subtypes of cell lines areIgG,IgM,IgG,IgM,respectively.And #1 and #7 were selected for further propagation as IgG subtype.The cell culture supernatant was collected for testing antibody specificity with Western Blotting(WB).Cow serum over 35 days of gestation,over-expression of PAG1 with HEK293 cells and PNGase F deglycosase were used to WB.Two positive hybridoma cells with good specificity were injected into the abdominal cavity of mice to collect ascites,then extract monoclonal antibodies from ascites and then purify them.After that,the double antibody sandwich ELISA was established by using the selected monoclonal antibody.After optimized,serum samples from day 15 th of AI and day 30 th of AI were collected and compared with the results of B-ultrasonography at 2 months after AI.After day15 th of AI,the positive coincidence rate was 75%,the negative coincidence rate was72.7%,and the total coincidence rate was 74.1%.After day 20 th of AI,the positive coincidence rate was 91.3%,the negative coincidence rate was 83.3%,and the total coincidence rate was 89.7%.