牦牛HMGB1多克隆抗体制备及应用OA

This study aimed to clone the HMGB1(high mobility group box chromosomal protein 1)gene of yaks(Bos grunniens),prepare polyclonal antibodies against the HMGB1 protein of yaks,and attempt to analyze its correlation with yak uterine health and reproductive status.The HMGB1 gene sequence was obtained from yaks through gene cloning;purification of yak HMGB1 recombinant protein by prokaryotic expression and affinity chromatography,preparation of yak HMGB1 polyclonal antibody by Ni column chromatography,and determination of antibody titer using enzyme-linked immunosorbent assay(ELISA);and real-time fluorescence quantitative PCR(qRT PCR)was used to compare the relative expression levels of HMGB1 in the inflamed uterus and uterus at different estrus stages.The expression levels and localization of HMGB1 protein in different yak uterus were detected using prepared antibodies.The experiment successfully cloned the yak HMGB1 gene(GenBank No:OR713743);The recombinant plasmid pET-28a-HMGB1 was constructed and successfully expressed recombinant proteins in both inclusion bodies and supernatant;The purified yak HMGB1 recombinant protein has a size of 25 kDa,which meets the expected results.After immunization with Japanese rabbits,the antibody titer was 1:128000 with good specificity.HMGB1 mRNA and protein detection found that the expression level of HMGB1 was highest in inflammatory uterine samples,followed by follicular and pregnancy uterine samples,and the lowest in luteal phase uterine samples.The experiment found that HMGB1 can serve as a warning factor for yak uterine inflammation,and its expression level in the uterus is related to yak estrus status.This provides a theoretical basis for exploring the involvement of HMGB1 in yak estrus regulation and uterine health.