Construction And Expression Of MUC1-VNTRs Gene Vaccine For Multiple Myeloma

Multiple myeloma,also known as myeloma or plasma cell myeloma,is a progressive hematologic disease.It is characterized by excessive numbers of abnormal plasma cells in the bone marrow.The peak age for multiple myeloma is among the elderly,and the average age at diagnosis is about 68 years.The median duration of survival was approximately 33 months in review of 1027 multiple myeloma patients in the United States in 2006,so multiple myeloma has poor prognosis.By far multiple myeloma is not considered curable with current approaches, and it is still considered the only incurable tumor in three major hematologic malignancies:leukemia,lymphoma and multiple myeloma.Although the therapy of multiple myeloma has got many progress,but most of myeloma patient may relapse, which make effective treatment limited,and in some extent greatly consume society and public health resources.For this reason,the importance of developing new treatment strategies for this currently incurable disease should be highlighted and will become very important need of current research project.MUC1(mucin1)is a tumor associated antigen(TAA)for multiple myeloma,which was aberrantly expressed in multiple myeloma cells,and MUC1 variable number tandem repeats(VNTRs) expressed in cell outer membrane contained many antigen epitopes,which could induce body producing specific immune response.MUC1 could induce body to activate CTL by MHC restricted and non-restricted ways,and then the activated CTLs would kill the multiple myeloma cells that have expressed MUC1 protein,so VNTRs is the best sequence to induce body to produce specific cytoimmunity and humoral immunity response.We used two tandem repeated sequence of extracellular MUC1 (2VNTR)as target antigen,and used its coding gene to construct eukaryotic expressing vector pcDNA3.1/MUC1-VNTRs/myc-his B as gene vaccine for multiple myeloma,then transfected into CHO cell lines to learn the expressing information about 2VNTR gene sequence by ELISA method.The constructed gene vaccine will establish the foundation for the further animal immunity experiment.Part OneThe construction and identification of MUC1-VNTRs Gene vaccine for multiple myelomaObjectiveUsing MUC1 extracellular two tandem repeats(2VNTR)which is a tumor associated antigen of multiple myeloma as target antigen to construct eukaryotic expression vector pcDNA3.1/MUC1-VNTRs/myc-his B as gene vaccine for multiple myeloma,transforming into competent cell JM109,culturing and selecting bacteria colonies to analyze the right fragment with restricted enzyme analysis,and then sending positive colony to biologic company for DNA sequencing.MethodsRedesigning 2VNTR coding gene as target gene,a COZAK sequence was inserted before 2VNTR,and two enzyme restriction sites were inserted:Hindâ…¢was in the front,and Xbaâ… was in the end.Then the recombinant gene of 2VNTR was synthesized and cloned into MCS in the pcDNA3.1/myc-his B plasmid vector,next the recombinant vector pcDNA3.1/MUC1-VNTRs/myc-his B was transformed into competent cells JM109,and grew the bacteria JM109 in LB plate containing ampicillin.Then selecting several colonies for restriction enzyme analysis,the right recombinant vector was sent for DNA sequencing. ResultsTwo colonies were selected to identify with restriction enzyme analysis.Among them,one colony could be seen a fragment between 100bp and 200bp.Then selecting the positive colony for DNA sequencing that showed recombinant pcDNA3.1/MUC1-VNTRs/myc-his B included whole reading frame and 2VNTR.ConclusionsThe constructed pcDNA3.1/MUC1-VNTRs/myc-his B for multiple myeloma has been proved the gene sequence was correct by using DNA sequencing.Part twoExternal transfection and expression of MUC1-VNTRs gene vaccine in CHOObjectiveGene vaccine means that the target gene of coding external antigen was inserted into a vector containing eukaryotic expressive system,and under the regulation of promoter,enhancer of eukaryotic expressive vector,the target gene could finish transcription and translation,and then expressed corresponding antigen proteins.The expressive proteins that were polypeptide or protein would be presented and bound to MHC,which would induce body to produce corresponding antibody and made CTL to proliferate in great amount,which processed body's humoral and cellular immune response.Whether the target gene should be expressed effectively in the body is the key point of gene vaccine immune effect.The experiment was that using the constructed vector pcDNA3.1/MUC1-VNTRs/myc-his B to transfect CHO cells to learn about the protein expression in CHO cells,and we could lay a foundation for further research about gene vaccine inducing mouse to produce specific CTL and antibody reaction..MethodsUsed the constructed vector pcDNA3.1/MUC1-VNTRs/myc-his B to prepare large amount of DNA,and transfected into CHO cell lines with GFP as reporting gene. After 48 hours,observed if the cells produced fluorescence signal,if do,continued growing cell until 2 weeks,and got the cell pellet to test the MUC1-VNTR peptide expression.ResultsTransfected the constructed pcDNA3.1/MUC1-VNTRs/myc-his B and original vector pcDNA3.1/myc-his B into CHO with GFP as reporting gene,after 48 hours, we could see the fluorescence signal in CHO,and after 2 weeks,we collected cell pellet and used ELISA method to test the MUC1-VNTR peptide,pcDNA3.1/myc-his B was used as negative control,and we got the result that the constructed vector pcDNA3.1/MUC1-VNTRs/myc-his B has got the MUC1-VNTR peptide expression, and the original vector pcDNA3.1/myc-his B has nothing.ConclusionsThe successful constructed vector pcDNA3.1/MUC1-VNTRs/myc-his B could express VNTR peptide in mammal cell lines,which offered the experimental basis to carry out the next animal immune research.