The Preparation And Detection Of Polyclonal Antibody Of Common Reference Genes In The Silkworm,Bombyx Mori

Reference genes are expressed in all the tissues and cells and are often used to study the expression and function of other genes and proteins.As a model organism of Lepidoptera insects,Bombyx mori plays an essential role in the study of insect genomics,gene regulation,molecular mechanism and genetic development.Reference genes play important roles in the study of gene function in silkworm.According to the special silkworm life cycle,the change of signal transduction,protein synthesis and metabolism of rapid and significant differentiation of specific tissues,the high functional differences between the larger organization,selection of suitable reference gene not only can be used for the analysis of gene and protein expression level in silkworm,but also for functional studies of genes in Lepidoptera and other insects.In this paper,the silkworm Bombyx mori is the research object,the most commonly used reference genes(gapdh,actin,tubulin)were cloned,expressed in prokaryotic system and purified.The antibodies were prepared and then measured the titer of each antibody.Western blot were further performed with the crude proteins from silkworm and other species in order to detect and compare the efficience of these antibodies to the commercial reference protein antibodies from mammals.The results showed that GAPDH,ACTIN and TUBULIN proteins were successfully expressed in prokaryotic cells,and the recombinant proteins were purified.The polyclonal antibodies were then prepared.The results of enzyme linked immunosorbent assay(ELISA)showed that the titer of all the three prepared antibodies titers was more than 8000.Western blot results indicated that each antibody has stong and specific hybridization band,they are not only specific to silkworm,but also to other insects and species.In summary,the prokaryotic expression vector of gapdh/pET-28 a,actin/pET-28 a,tubulin/pET-28 a,were constructed.Their recombinant proteins were expressed and purified.The polyclonal antibody of each protein was prepared by immuning rabbit.and the titer,specificity in silkworm and other insects and species were further analysed.This work provides a foundation for further studying the function of other genes and proteins in lepidopteran insects and other species,meanwhile,it has important scientific significance and broad application prospect.