Polyclonal Antibody Production And Expression Pattern Analysis Of Buffalo Maternal Effect-genes NLRP5?TLE6 And PADI6

It is well-known that the NLRP5,TLE6 and PADI6 genes belong to the members of the subcortical maternal complex(SCMC),which play key roles in oocyte growth and the development of early embryos before implantation.The study of maternal genes is mainly foused on human and mice,less aware of large domestic animals such as water buffalo,and few reports on regulation mechanism of expression patterns of maternal gene in large mammals.In addition,there is a lack of antibody products of buffalo maternal genes in the market of biological products,so it is difficult to conduct in-depth research on their function and localization and other mechanisms.Therefore,the preparation of polyclonal antibodies against NLRP5,TLE6 and PADI6 in buffalo and further study of their expression patterns will provide theoretical guidance for understanding the growth of buffalo oocytes and the mechanism of early embryo development before implantation.In this study,the three buffalo maternal genes were selected as research objects,and gene cloning,polyclonal antibody preparation and their expression patterns were studied respectively.The results are as follows:1.The complete sequence of NLRP5 gene coding region(CDS)3297 bp was successfully cloned,encoding 1098 amino acids,and the predicted molecular weight was 121.17 k D.The full-length sequence of the coding region of buffalo TLE6 gene 1731 bp was successfully cloned,encoding 576 amino acids,and the predicted molecular weight was 64.09 k D.The 2121 bp fragment of coding region sequence of PADI6 gene in buffalo was cloned,which was still missing 177 bp compared with 2298 bp,and the predicted molecular weight was 85.35 k D.Sequence analysis showed that the nucleotide sequences of the cloned three genes had a high homology with that of cattle,goats and sheep,indicating that the three genes were conserved in evolution among different species.The structure and function of NLRP5,TLE6 and PADI6 proteins were predicted,suggesting that NLRP5 and TLE6 may be related to the interaction between proteins in buffalo.2.The prokaryotic expression vectors of NLRP5,TLE6 and PADI6 in buffalo were constructed and their polyclonal antibodies were prepared.Kunming mice were immunized with purified recombinant proteins to produce polyclonal antibodies.Western blotting was used to detect the titers of antibodies and recombinant proteins,and the results showed that the titers of polyclonal antibodies of the above three genes reached 1: 64 000,1: 64 000 and 1: 52 000 respectively,indicating that the titers of polyclonal antibodies were relatively high.3.The expression patterns of NLRP5,TLE6 and PADI6 genes in buffalo oocytes and parthenogenetic activated early embryos were detected by real-time fluorescence quantitative PCR.The results showed that the transcription was largely expressed in the oocytes of MII stage,and decreased with the continuous cleavage,and almost no expression was detected in the morula and blastula stages.The results of semi-quantitative PCR showed that NLRP5 was highly expressed in oocytes and slightly expressed in ovaries and testis,while TLE6 and PADI6 were only expressed in oocytes.The expression patterns of the three proteins in buffalo tissues were analyzed by Western blotting,and the results showed that they were only expressed in oocytes.Immunofluorescence detection of the expression patterns of NLRP5,TLE6 and PADI6 proteins in oocytes during maturation and in early embryos at each stage showed that the three proteins were present throughout the maturation of buffalo oocytes and the development of early embryos to the blastocyst stage.In conclusion,we constructed the prokaryotic expression vectors of buffalo NLRP5 gene(480 bp),TLE6 gene(198 bp)and PADI6 gene(504 bp)and successfully prepared three polyclonal antibodies with good specificity.At the same time,the expression patterns of three maternal genes and their corresponding proteins were detected.This experiment results will lay the foundation for further study of the mechanism of oocytes growth and early embryonic development in buffalo.