Expression And Biological Activity Of Staphylococcal Enterotoxin E & Immobilization Of Human Genomic DNA

â… .Expression and Biological Activity of Staphylococcal Enterotoxin E Dr.White J.proposed the conception of superantigen in 1989:it was a kind of proteins that could stimulate a large number of T cells at an extremely low concentration.Due to its most potent activities on T lymphocytes,it was named as superantigen.And staphylococcal enterotoxins belonged to the most well studied typical superantigens.Different from other common antigens,they required only recognition of specific TCR Vβfor interaction and cross-link the T-cell receptor and antigen-presenting cells of MHC classâ…¡,initiating activation.Until recently,about 20 kinds of SEs were found,including SEA～SEE,SEG～SEQ and TSST-1.SEA,SEB and SEC2 belonged to the most well studied group among all the SE serum types,while other serum types were ignored to some extent.As a novel type of staphylococcal enterotoxin,SEE owned higher thermal stability and much lower seroactivity and immunogenicity than other SEs.In order to obtain pure SEE and lay the way for intensive research on SEE anti-tumor mechanism,we tried to harvest recombinant protein through genetic engineering.First of all,see was amplified through PCR from S.aureus,cloned into expression plasmid and then transformed into competent E.coli BL21 for induced chemical expression. After affinity chromatography and ion-exchange chromatography,rSEE was obtained at the purity of 85%.MTT results indicated that rSEE's strong ability to stimulate mice lymphocyte proliferation,which was a dose-dependent effect.Through the proliferation of mice splenic lymphocyte,rSEE had a strong lethal effect on tumor cells K562-AD,K562 and B16.Purified rSEE with a superantigen activity would provide bedrock for the study on anti-tumor mechanism of rSEE and construction of targeted anti-tumor fusion protein.1.Cloning and sequencing of see from S.aureus FRI 326Primers were designed according to the DNA sequence of SEE from GenBank (M21319).Recognition site of BamH I was introduced at the 5'terminus of the forward primer,meanwhile a stop codon and recognition site of Xho I was introduced to the 5' terminus of the reverse primer during automated oligonucleotide synthesis. Extraction of genome DNA from S.aureus and PCR amplification were performed. After the retrieve and cloning of target gene segment into the vector pUCm-T,DNA sequencing was performed to verify the positive clone with correct sequence inserted.2.Construction of recombinant pGEX-4T-1-SEE expression plasmidThe correct target segment after digestion with BamH I and Xho I was subcloned into linear expression plasmid pGEX-4T-1.And the product was transformed into competent E.coli DH5αfor subsequent sequencing.Then the recombinant plasmid pGEX-4T-1-SEE with correct sequence was transformed into competent BL21(DE3) cells.3.Induced expression of recombinant strain and solubility analysisE.coli BL21(DE3)transformed with pGEX-4T-1-SEE was cultured overnight at 37℃in 2×YT(Amp 100 mg·L^-1).The culture was injected into fresh 2×YT with the inoculation quantity of 1%,cultured at 30℃for 3h,IPTG(0.1 mmol·L^-1)added for overnight induction.The result of SDS-PAGE indicated that the soluble target protein occupied 40%germ protein in soluble form.4.Purification of fusion proteinThe GST-tag in the fusion protein allowed purification through glutathione Sepharose 4B.The GST-SEE protein was eluted down by the reduced-glutathione.A thrombin digestion-site existed between the sequence of GST and SEE.After digestion with thrombin at 16℃for 16 h,ultra-filtration was applied for desalting from rSEE mixture.Then ion-exchange chromatography was performed for further purification.5.HPLC analysis of rSEEEquipment:Agilent 1100 Series HPLC system;chromatography column:Bio-rad Bio-Sil SEC 250-5(300mm×7.8mm);mobile phase:TEAN buffer(pH 7.0);flow rate: 0.5 mL·min-1;Detection wavelength:280 nm;injection volumn:100μL.6.Biological activity assay of rSEEWe utilized classic MTT method for the analysis of rSEE's biological activity.By observing the activation and the lethal effects on tumor cells of mice lymphocyte, activities of rSEE were tested.K562,K562-AD and B16 were utilized as the operated cells.The results indicated strong activities of rSEE to stimulate mice lymphocyte proliferation,which was a dose-dependent effect.With the proliferation of mice splenic lymphocyte,rSEE had a strong lethal effect on tumor cells K562-AD,K562 and B16.In all,rSEE at the purity of 85%provided the basis for the preparation of monoclonal antibody and polycionai antibody of SEE.MTT method indicated the classic characteristic of superantigen of rSEE:splenic lymphocyte proliferation stimulation and tumor cell inhibition at relatively low concentration,which provided the bedrock for further research on the SEE's anti-tumor mechanism and construction of targeted anti-tumor fusion protein. â…¡.Immobilization of Human Genomic DNACould we assume an individual genome keep identical during the lifetime without regarding gene mutations caused by the attack of exogenous and endogenous DNA damaging substance? Could we present a comparative analysis for functional genomics without regarding the inheritable mutation induced by arrest of the replication fork,and/or the formation of replication gaps during DNA replication?In fact,the integrity of our genetic material is being challenged due to the exposure of cells to exogenous chemicals constantly(e.g.environmental agents,food constituents, etc.).Even without excessive exposure to DNA damaging chemicals,estimates have predicted that each day genomic DNA suffers more than 20,000 hits due to endogenous cellular metabolite.While DNA damage is the major cause of mutation, and it has been estimated that spontaneous mutations arise in about a third of cells divided per second.Despite the presence of error-free DNA repair mechanisms,a large number of DNA lesions escape from repair mechanisms.These lesions might evoke arrest of the replication fork,and/or the formation of replication gaps during DNA replication,which could be processed to complete replication and enable cell division.Since,people began to realize genetic changes during disease and lifespan, there is an increasing need for ascertaining the dynamic changes occurring in the genome.And that might provide a better understanding of pathogenesis,diagnosis, prevention and treatment of common diseases.As for cancer,it is now believed that cancer results from cumulative genetic damages in a susceptible cell.Basically,the genetic damage is mainly due to somatic mutations but may also involve germline mutations.Although people attempt to figure out the mechanism and pathway of DNA damage during the tumorigenesis,little advancement in understanding the detailed occurrence and potential impact,is obtained.To solve this problem,it is first important to distinguish between the genetic basis,causal genetic changes that produce oncogenes with dominant gain of function and tumor suppressor genes with recessive loss of function and the bystander genetic changes,hallmarks of cancer,that accumulate during pathological developments.Limited by technical barriers,systematic comparative analysis on genetic alteration during the diseases is neglected.With the advancement in sequencing and genotyping technologies,it is clear that both detection on SNP sites of all putative causal variants and whole genome sequencing are optional approaches to study the dynamic transformation contributing to disease.The sequencing and genotyping technologies are still not fully developed,but it is obvious that there exist substantial benefits from having a person's whole genome available for current and future testing needs rather than waiting until technologies are fully developed to obtain and deal with the information contained in the double helix.Once,the ability to predict the onset of disease grows significantly,prevention of the rate-limiting,causal mutations to impede the advancement of disease will become available.Although,the collection and store of genomic DNA before disease onset have been on process,due to prospective cohort studies aimed at identifying risk factors that predispose an individual to disease,or biomarkers for predicting disease development,it would be much easier for understanding the health condition,predicting disease,taking preventive action and giving personalized treatment accordingly to keep genetic information from infancy to death at hand as individual medical records.Considering millions of potential mutations related to diseases and limit of DNA yield from human specimens,DNA usage should be minimized so that millions of SNPs can be interrogated using a relatively small amount of genomic DNA.Several high-throughput platforms for SNP genotyping have already achieved the goal of miniaturization(0.16 ng DNA/genotype),but comparative analysis on all putative SNPs still requires a huge amount of DNA.And,permanent genome immobilization seems to be an ideal alternative and/or supplement to realize retrieve and effective utilization of the double helix for future data acquisition.So,in order to realize retrieve and effective utilization of human genomic DNA,we applied the DNA-hydrogel copolymerization chemistry to immobilize DNA on the substrate.After the anaylsis on the efficiency and thermal stability of this attachment chemistry,this strategy was utilized for the attachment of plasmid DNA and human genomic DNA successfully.1.Thermal stability of DNA-hydrogel copolymerization chemistryPrimers with amines at 5' terminals were designed according to the DNA sequence of pUCm-T-SEE.Amine-labeled 850bp SEE DNA fragment(NH₂-T7-SEE- Puc26- NH₂) was generated by PCR amplification with primer T7-amino and Puc26-amino.After the purification of PCR product,it was diluted in distilled water.With the catalyzation of TEMED and AP,the amine of PCR product acted with methacrylamide and was immobilized on the hydrogel.PCR reactions were performed to check the status of DNA immobilization.2.Immobilization of plasmid DNAAfter partial depurination of pUCm-T-SEE,the generation of activated aldehyde eased subsequent reaction with ethylene diamine.In this way,amino group was introduced into DNA for further copolymerization.The similar procedure as mentioned above was utilized to check the feasibility of this strategy for immobilizing plasmid DNA on hydrogel for PCR reaction.3.Immobilization of human genomic DNAAfter partial depurination of human genomic DNA,the generation of activated aldehyde eased subsequent reaction with ethylene diamine.In this way,amino group was introduced into DNA for further copolymerization.The similar procedure as mentioned above was utilized to check the feasibility of this strategy for the attachement of whole genome on hydrogel for PCR reaction.In all,it is our idea that DNA samples are valuable resources especially for the exposure of influencing factors related with diseases,discovery of novel biomarkers, prediction of disease,and guidance for effective treatment.It is obvious that there exist substantial benefits from having a person's whole genome available for current and future testing needs,rather than waiting until technologies are fully developed to obtain and deal with the information contained in the double helix.So,the database of individual genome will serve as bedrock for self-comparative analysis of human genome,and play an important role in a future world of fast diagnosis of diseases & individual medicine.In this study,we selected an appropriate chemical method for DNA immobilization on hydrogel for solid phase PCR.We evaluated the immobilization efficiency and thermal stability of this attachment chemistry using PCR product.By optimizing the parameters,we tried to utilize this strategy to chemically attach fragmented plasmid DNA and fragmented human genomic DNA on hydrogel as templates for subsequent PCR reactions.And the preliminary conclusion indicated a bright future of this technology for whole genome sequencing and SNP detection,and it opened the door for systematic self-comparative analysis at genome level during lifespan.