Cloning And Prokaryotic Expression Of Acid Protease Gene And Preparation Of Polyclonal Antibody Of Inonotus Obliquus

Inonotus obliquus is a very rare and valuable wild medicinal fungi,the medicinal part of the sclerotium.At present,although many scholars have successfully cultivated inoculation of Inonotus obliquus,and can form sclerotia,but its biological efficiency is too low,seriously limiting the large-scale production of Inonots obliquus.In order to study the factors that influence the yield of sclerotia from the molecular level,the bioinformatics analysis of the gene was carried out for the first time by RACE technique,the gene was analyzed by bioinformatics,and the gene was prokaryotic expression and polyclonal antibody preparation.The results are shown blow:1.The full-length cDNA of IO-AP was cloned from the strain Inonotus obliquus JL01 by degenerate PCR and RACE.The full-length sequence of IO-AP gene was 1178 bp,encoding 329 amino acids.2.Encoding the amino acid sequences of IO-AP gene were carried out on similarity search,the results show that the IO-AP hace high identity with other gungal AP,IO-AP protein has the highest identity with the AP of Sanghuangporus baumii which is 88%,followed by the coincidence of other fungal AP proteins,further indicating that the cloned gene sequence is the target gene sequence.3.The results shown that IO-AP protein is closest relative with Hymenochaetaceae Sanghuangporus baumii by constructing the phylogenetic tree,indicating that there is a certain correlation between the evolutionary relationship between proteins and the evolutionary relationship of species.4.Bioinformatics analysis showed that the IO-AP protein has not signal peptide and belongs to the stable protein,which exists in the free state of the cell.The protein belongs to the family of aspartic protease A1 of the pepsin super family and belongs to the acidic protease.Its biological function is involved in protein hydrolysis,with aspartic acid type endoprotease activity.5.In this study,the prokaryotic expression vector pGEX-4T1-IO-AP was constructed successfully.Stable expression in E.coli BL21(DE),and in the form of inclusion bodies,the molecular weight of recombinant protein is about 60 KDa.6.In this study,we used the method of recombinant expression of IO-AP in prokaryotic cells and obtained 4 Balb/c mice immunized with IO-AP as immunogen.The immunization process was performed by traditional 3-2-2-2 immunization method,IO-AP as antigens,by indirect ELISA method to determine the antiserum titer,after the fourth immunization,4 mice antiserum titers were greater than 121500.7.In this study,the preparation of IO-AP antiserum prepared by the initial detection of recombinant protein IO-AP ELISA method,through the examination of the board to determine the best antigen concentration of 1 μg/mL,antiserum best dilution ratio of 1:4000.8.The recombinant protein was detected,and the recombinant protein(5 ng)was detected.The results showed that the preparation of IO-AP polyclonal antibody was successful.