Eukaryotic Expression Vector Construction, Expression And Biological Activity Identification Of HLA-B27Antagonistic Peptide

Ankylosing spondylitis (AS) is a chronic systemic autoimmune disease, itseriously endanger the human health and quality of life, and seriously affect theworkforce, its insidious onset, longer duration, hard to be cured, eventually leadingtopatients with loss of activity, even disabling. And because the pathogenesis of thedisease is not yet clear until now, there is no effective cure cure for ankylosingspondylitis. Therefore, the exploration of the AS treatment strategies is always thehot and difficulty focus of studies of particular concern at home and abroad, Atpresent, the use of drugs and programs are mainly in still non‐specific relievesymptomatic therapy, this will produce a variety of more or less serious side effects.Therefore, how to create the new drug with low toxicity and high specificity, whichcan quickly relieve the symptoms of AS, but also can effectively selective blockade ofthe pathophysiology of AS has become a key problem to be solved in the field ofRheumatology immunology!In theory, anything of the drug that can effectively inhibit or block ASpathophysiology and development process can effectively reduce or alleviate the ASin bone and joint disease. In view of the fact that HLA‐B27is the central link in theimmune pathological process of the development of AS, selectively block thesignaling pathways of the HLA‐B27became one of the important strategy foreffective treatment of AS autoimmune diseases. Therefore, this project intends fullyorganic combination of the two strategies: block the immune and gene therapyvaccines, innovative development of the new type of treatment based on blockingthe signaling pathways of the HLA‐B27gene vaccine. Develop the new type oftherapeutic gene vaccine which canblock the HLA‐B27signaling pathway. In this study, we use the preliminary work in the use of HLA‐B*2704and HLA‐B*2705heavychain extracellular domain phage12‐mer random peptide and obtaine its specificcombination of antagonistic peptide (AP).The main purpose of this study is to therapeutic vaccine of AS: HLA‐B*2705–AP.Construct the eukaryotic expression vector, and expression in vitro identification,and preliminary study on its biological activity. In this study, weidentified theConstruction and expression of eukaryotic expression vector of pcDNA3.1‐B*2705‐AP,and a preliminary study of its biological activity. However, due to the HLA‐B27antagonistic peptide molecular weight is too small, is not conducive to in vitroidentification and observation. Therefore, this study further to build a fusion ofenhanced green fluorescent protein coding genesconstruct the fusion expressionvector pcDNA3.1/HLA‐B*2704‐AP‐EGFP and the fusionexpression vectorpcDNA3.1/HLA‐B*2705‐AP‐EGFP. Through a series of studies to obtain the followingresults:Part1:The construction, expression of pcDNA3.1/B*2705‐AP eukaryoticexpression vector and the preliminary study of biological activity1.We obtained the gene of HLA‐B*2705‐AP by polymerase chain reaction(PCR),gained the reactions of the PCR were carried out for30cycles at98℃for10s,60℃for15s,72°C for30s. The end of the cycle and then72℃for8minutes,and finally stored at4℃.144bp HLA‐B*2705‐AP was assemblied by PCR.2.We use T4ligase connected HLA‐B*2705‐AP gene with pMD18‐T vector for cloningin E. coli Top10, PCR result and sequencing results showed that the target gene isalready connected to the carrier.3.Digest the pMD18‐T/HLA‐B*2705‐AP vector and pcDNA3.1（+）‐EGFP vector withKpn Ⅰ and Xba Ⅰ, construct the recombinant vector of pcDNA3.1(+)/HLA‐B*2705‐AP cloned in Ecoli Top10. 4.We transfect cos7cells with the recombinant expression vector pcDNA3.1(+)/HLA‐B*2705‐AP by GenFectin mediated method.We use RT‐PCR detect theexpression of B*2705antagonistic peptide in Eukaryotic cells. Electrophoresisshowed that cells transfected with the eukaryotic expression vector specific bandsvisible at100bp, without the transfected cells did not.5.Through the complement‐dependent HLA‐B27positive lymphocyte cytotoxicity test,the supernatant of the transfected eukaryotic cells can effectively block the killingeffect of the specificity of HLA‐B27.Part2:The construction, expression of pcDNA3.1/B*2705/2704‐AP‐EGFPeukaryotic expression fusion vector and the preliminary study of biological activity1.We obtained the gene of HLA‐B*2705‐AP and HLA‐B*2704‐AP by polymerase chainreaction (PCR),gained the reactions of the PCR were carried out for30cycles at98℃for10s,60℃for15s,72°C for30s. The end of the cycle and then72℃for8minutes, and finally stored at4℃.144HLA‐B*2705‐APwas assembliedby PCR.2.Digest the pMD18‐T/HLA‐B*2705‐AP/B*2704‐AP vector and pcDNA3.1（+）‐EGFPvector with Kpn Ⅰ and Xba Ⅰ, construct the recombinant vector of pcDNA3.1(+)/HLA‐B*2705AP/B*2704‐AP‐EGFP cloned in Ecoli Top10.3. We transfect CHO cells with the recombinant expression vector pcDNA3.1(+)/HLA‐B*2705AP/B*2704‐AP‐EGFP by GenFectin mediated method.Underfluorescence microscope can clearly see the green fluorescent protein expressed bycells in the flow results showed that the proportion of fluorescent cells to total cellsis64.86%and57.91%, respectively, indicating higher transfection efficiency.4.We use RT‐PCR detect the expression of B*2705-AP-EGFP andB*2704-AP-EGFPin Eukaryotic cells. Electrophoresis showed that cells transfected with the eukaryoticexpression vector specific bands visible at1000bp(Sequencing primers CMV‐f andBGH‐r), without the transfected cells did not. This shows that the cells haveexpression the mRNA of the fusion protein.5.We use Western blot detect the fusion protein antigen. The results showed that thecells transfected with the recombinant vector relative molecular mass ofapproximately30kD positive bands, empty vector‐transfected cells display of anystripe.6.In the experiment of antiserum against HLA‐B27mediated complement‐dependentHLA‐B27positive lymphocyte cytotoxicity,100%will cell death. Add5μl disrupted cellsupernatant which transfection of the recombinant plasmid B*2705‐AP‐EGFP and B*2704‐AP‐EGFPto this experimental system, respectively,50%and40%of thecytotoxic response was blocked, indicating recombinant proteins expressedspecifically block the B27reaction.7.Using fluorescence microscopy examinethe binding of AP and cell, B*2705‐AP‐EGFPand B*2704‐AP‐EGFP do not bind with HLA‐B27negative B cells, but can becombined with HLA‐B*2705and B*2704positive cells, show that this kind ofcombination with specificity, at the same time with the method of FC detection isfurther proof that the specific binding.In summary, this study successfully constructed the eukaryotic expression vectorof the HLA‐B*2705AP and through gene transcription, protein expression, specifictests and preliminary study of the biological activity,provides solid experimental dataand theoretical support to the depth study of the the HLA‐B27AP AS therapeuticDNA vaccine.