The Application Of Pregnancy-Associated Glycoproteins In Early Pregnancy Diagnosis Of Dairy Cows

Pregnancy-associated glycoproteins(PAGs),a large family of glycoproteins,are synthesized and secreted by trophoblast cells,which play important roles during pregnancy in cow species.PAGs measurement in body fluids has become one of the most efficient methods of early pregnancy diagnosis,and has important significance in improving reproductive efficiency and economic benefits in the farm.Therefore,this study aimed to investigate the accuracy of PAGs detection kits,isolation and identification of PAGs,as well as prokaryotic expression of PAG4 gene and preparation of PAG4 polyclonal antibody,in order to provide technical supports for developing PAGs detection products.The results were as follows:Experiment I: Application of PAGs Detection Kit in Early Pregnancy Diagnosis of Dairy CowsPAGs detection kit was used to detect the concentration of PAGs in blood samples collected from49 cows subjected to artificial insemination(AI).Pregnancy diagnosis was simultaneously performed by ultrasound examination(veterinary B-mode ultrasound scanner)on the sample collection day,and re-examined at Day 35 after AI to further confirm the pregnancy diagnosis by B-ultrasound.The results showed that: The sensitivity,specificity,positive predictive value,negative predictive value,and the accuracy of PAGs detection kit were 92.0%,95.8%,95.8%,92.0%,and 93.9%,respectively.The statistical agreement(kappa value)between PAGs detection kit and B-ultrasound assay was 0.632.Experiment II: Isolation and Identification of PAGs in Dairy CowsFour cow fetal placentas were collected at local abattoirs.Saturated ammonium sulfate precipitation,anion chromatography,SDS-PAGE,liquid chromatograph-mass spectrometric(LC-MS)were used to isolate and purify PAGs.The results showed that: The relative molecular mass of the purified protein was about 55 kD;Eight members of PAGs family were successfully identified,including PAG1,PAG2,PAG6,PAG7,PAG9,PAG12,PAG13 and PAG22.Experiment III: Prokaryotic Expression of PAG4 Gene and Preparation of Polyclonal AntibodyThe target fragment of PAG4 gene,amplified in vitro,was cloned into pET28 a vector.The recombinant vector was transformed into E.coli DH5 a,and confirmed by PCR and sequencing.The correctly identified plasmid was transformed into E.coli BL21.PAG4 recombinant protein was expressed after induction by IPTG.The polyclonal antibody was prepared by immunizing New Zealand white rabbit with the purified PAG4 recombinant protein.Antibody titers were detected by indirect ELISA and antibody activities were measured by western blot.The results showed that: The prokaryotic expression vector of PAG4 polypeptide and the purified PAG4 recombinant protein were successfully obtained.The titer of the polyclonal antibody was up to 1: 24,000,and the acquired polyclonal antibody could specially react with the recombinant and native proteins.In addition,this polyclonal antibody could determine whether a cow was pregnant or not.