Expression Of Human Recombination Activity Gene-1

Objective:Recombination activating genes(RAGs) are essential for catalyzing the recombination response, which consists of two genes : RAG-1 and RAG-2. The products of RAG-1 and RAG-2 protein complex, an important recombinase, initiate V(D)J recombination by introducing DNA breaks at Recombination Signal Sequences(RSS) flanking a pair of antigen receptor gene segments. V(D)J joining is a site-specific recombination process that plays a crucial role in the activation and diversification of antigen receptor genes. Mutations or aberrant expression of either RAG-1 or RAG-2 in human completely prevented the rearrangement of immunoglobulin (Ig) and T cell receptor (TCR) genes blocked the development of mature B and T lymphocytes and cause combined immunodeficiency. Recently, with the development of molecular biology technique and molecular mechanism investigation of genetic disease, the study of gene therapy makes greet progress. Research object in this experiment is to clone the complete coding sequence of RAG-1 gene and construct its eukaryotic expression vector, in order to provide experimental evidence for sufficient expression and further functional study of RAG-1, which will contribute greatly to the gene therapy of human RAG-SCID.Method:1. RNA isolation: Total cellular RNA was extracted from human Jurkat cell lines with RNAiso Reagent. The purity of mRNA was confirmed by Oligotex-dT30 kit, according to the manufacturer's protocol.2. RAG-1 cDNA amplification:The cDNA fragment coding for RAG-1 gene was amplified by reverse transcription polymerase chain reaction (RT-PCR) from human Jurkat cells using Taq polymerase, two primers specific for the RAG-1 gene were used based on the published cDNA sequence of human RAG-1.3. Subcloning of human RAG-1 DNA: The purified PCR products were ligated with pMD19-T simple vector forming a new plasmid pMD-hR1CS, and transformed to the E.coli strain JM109 on LB/agar plates with ampicillin, the positive recombinant clones were further amplified.4. Construction of eukaryotic expression vector: The region of pMD-hR1CS containing RAG-1 gene was digested with BamH I and Xba I, detected by electrophoresis, purified and ligated by T4 DNA ligase, inserted into pcDNA3.1(+)vector, which resulted in the eukaryotic expression plasmid phR1CS.5. Detection of mRNA expression of recombinant RAG-1 in cells:Human 293T cells were transfected with phR1CS recombinant by SuperFect Transfect Reagent; all the procedures were performed according to the guidance of the reagent. To demonstrate the expression of RAG-1 mediated by phR1CS recombinant, total cellular RNA was extracted from the 293T cells. Synthesis and amplification of RAG-1 mRNA were performed using RT-PCR with specific primers for RAG-1.Result:1. Subcloning of human RAG-1 gene: The complete coding sequence of RAG-1 gene was amplified from human T cells total RNA by reverse transcriptase-polymerase chain reaction (RT-PCR). The PCR product showed a single band about 3219bp on 1% agarose gel. The recombinant of pMD-hR1CS was constructed with the human RAG-1 coding region inserted into the cloning site of pMD19-T Simple vector.2. Construction and sequencing of RAG-1 eukaryotic expression vector: A expected 3219bp fragment and 5366bp pcDNA3.1(+) fragment was obtained by restriction enzyme digestion from the recombinant plasmid phR1CS. The Positive clone sequence was consistent with the reported GenBank RAG-1 gene sequence (Accession number: NM000448). 3. Expression of recombinant RAG-1 gene in human cells: 293T cells were transfected with the recombinant plasmid phR1CS, cultured 40 hours, and cells were collected. RT-PCR analysis showed that 782bp fragments corresponding to the human RAG-1 cDNA were amplified with the total cellular RNA from phR1CS -transduced 293T cells.Discussion:In vertebrates the antigen binding domains of immunoglobulin (Ig) and T cell receptor (TCR) molecules are assembled from numerous variable (V), joining (J) and sometimes diversity (D) gene segments that are separated in the germline. These gene segments are assembled in a random, yet site-specific manner during early lymphoid development by a process called V(D)J recombination, which generates the diversity of antibody and TCR molecules. The complicated process of V(D)J recombination consists of two major processes: site-specific cleavage and ligation of cleaved ends. The former process includes specific recognition of the RSS by DNA-binding components of the recombinase and site-specific cleavage of RSS. The latter process is known to be mediated by nonhomologous end-joining machinery(NHEJ). The recombination activating proteins RAG-1 and RAG-2 directly participate in the recombination reaction, operate together to activate V(D)J recombination and thus they are both essential for this process. V(D)J recombination is important for the genesis of surface marker, function and texture of lymphocytes. The failure of functional expression of RAGs causes a defect in the formation of the functional antigen receptor of lymphocytes, and hence causes the block of lymphocyte differentiation and development in humans. It has also been shown that the missense mutations and abnormal expression of human RAG-1 or RAG-2 caused severe combined immunodeficiency without B lymphocytes and Omenn syndrome with a few antigen repertoires of lymphocytes. Currently, the treatment of choice is allogeneic hematopoietic stem cell transplantation (HSCT). However, in the absence of an HLA-identical donor, HSCT provides partially unsatisfying results.The successful construction of RAG-1 eukaryotic expression vector set a basis for available and sufficient expression of RAG-1; However, further studies will be required to evaluate biological activities of RAG-1 gene recombinant. Recently, with the development of molecular biological technique and study of molecular mechanism of hereditary disease, gene therapy gains a great progress; gene therapy, because of its stability, persistence, security and adaptability, would be an applicable therapeutic method. Successful application of therapeutic approach justifies this gene-therapy trial of human RAG-SCID, endorsed by results obtained in gene therapy of ADA- SCID patient. Our study set the basis for retrieving expression of RAGs gene and differentiation and development of lymphocyte by gene therapy and opens the way to radical cure of human RAG-SCID.Conclusion:Human RAG-1 coding region was successfully cloned by reverse transcriptase-polymerase chain reaction (RT-PCR), and its eukaryotic expression vector was constructed. Expression of mRNA in the recombinant phR1CS-transduced 293T cells was determined. The present study provides experimental data for sufficient expression and further functional study of RAG-1, which will contribute greatly to the gene therapy of RAG-SCID.