Preparation Of Polyclonal Antibody Against HSPA5 And Its Application In The Study On Drug Resistance In Non-Small Cell Lung Cancer

Heat shock protein A5 is a member of the HSP70 family,which promotes the correct folding of proteins,maintains the stability of the endoplasmic reticulum,and maintains the homeostasis of cells.However,recent studies have found that abnormally expressed HSPA5 is highly correlated with tumorigenesis,progression,metastasis,and drug resistance.Compounds,peptides and antibody targeting HSPA5 can inhibit tumor cell growth,moreover antibody drugs against HSPA5 have found specific ability of inhibit tumors in vitro and in vivo,and have achieved initial success in clinical trials.Furthermore,when searching for literatures related to TRAIL,it was found that endoplasmic reticulum stress and heat shock protein overexpression were the possible causes of drug resistance.In summary,we can speculate that the preparation of HSPA5 antibodies can be used to further explore the role of HSPA5 in tumor resistance and provide a possible drug strategy.Based on this,this paper based on the GEO database for a series of bioinformatics analysis of the data set numbered GSE55859,including differential gene,GO enrichment,KEGG enriches annotation,PPI analysis.Finally we speculates that HSPA5 may be a hub gene involved of TRAIL resistance in NCI-H460 cells.Subsequently,using the database such as Expasy to predict the partial properties of HSPA5 protein,the antigen expression and preparation strategy was proposed.Two HSPA5 coding genes were amplified by PCR,and then the genes were inserted into pET-28a and pGEX-6p1 vectors by DNA recombination technology.Two HSPA5 expression vectors were constructed:BL21/pET28a-HSPA5 and BL21/pGEX-6p1-HSPA5,and they were verified by colony PCR and double enzyme digestion.After identification of the sequence was corrected,IPTG was used to induce the expression of two fusion proteins,His-HSPA5 and GST-HSPA5,and we optimized the three conditions of IPTG induction:concentration of IPTG,induction temperature and induction time,further improved the protein expression efficiency;then use lysozyme to lysis BL21/pET28a-HSPA5 and BL21/pGEX-6p1-HSPA5,and two fusion proteins were separated and purified by sonication,ammonium sulfate precipitation and affinity chromatography,Subsequent we use BCA,SDS-PAGE,Western-Blot to identify the protein by molecular weight,purity,concentration and immunological properties.The molecular weights of the two proteins were in line with the theoretical value,the purity was greater than 90%,the concentration met the requirements of subsequent experiments,and the antigenicity of HSPA5 was retained.Thereafter,the His-HSPA5 fusion protein was used as an antigen,and New Zealand rabbits were immunized three times with Freund's adjuvant,and HSPA5 antiserum was obtained that having a titer of 1:10-5.HSPA5 polyclonal antibody was obtained by protein A affinity purification.The purity,titer and specificity of the antibody were determined by SDS-PAGE gel electrophoresis,ELISA and Western-Blot.,the result was that the antibody had a purity of more than 95%,a titer of 1:10-4,and good specificity.By comparing the commercially available HSPA5 antibodies,it was found that the HSPA5 polyclonal antibody was not significantly different from the commercially available HSPA5 antibody in Western Blot application.Finally,the activity of HSPA5 polyclonal antibody in NCI-H460 cells was detected by CCK-8.It was found that HSPA5 antibody could inhibited the growth of NCI-H460 cells,and the HSPA5 antibody could reduce resistance of TRAIL in NCI-H460 cells,which has certain application value.Through bioinformatic data analysis,this paper speculates that HSPA5 plays an important role in the resistance of NCI-H460 to TRAIL.Two HSPA5 soluble expression systems were subsequently established and a large number of HSPA5 polyclonal antibodies were obtained by immunizing New Zealand rabbits.This study demonstrates that HSPA5 is involved in the resistance of TRAIL in NCI-H460 cells,and demonstrates that the combination of HSPA5 antibody can reduce the resistance of NCI-H460 cells to TRAIL,which lays an important theoretical foundation for the follow-up study of HSPA5 as a drug resistance gene and provide indispensable experimental materials.