Construction And Identification A Recombinant Adenoviral Shuttle Plasmid Expressing BCL-2/IgH Gene Of Follicular Lymphoma

Objective:To construct recombinant adenovirus type 5(r Ad5)of shuttle plasmid carrying human BCL-2/Ig H gene,the purpose of this study is to make well preparation for expressing BCL-2/Ig H gene of r Ad.Methods:Total RNA was extracted from Do HH2 cell by Trizol method for amplification of BCL-2/Ig H fusion gene using RT-PCR. The segment of BCL-2/Ig H fusion gene was obtained by sequencing. After the blast of NCBI, the BCL-2 and Ig H gene sequences of Genebank were searched, and then CDS(Coding sequence) region of BCL-2 gene was determined. BCL-2/Ig H c DNA base on the Genebank was chemically synthesized and cloned into the plasmid p UC57-Amp.The plasmid was digested with Hindâ…¢ and Salâ… ,and the double-digested fragment was the cloned into adenovirtal shuttle plasid p DC316,forning p DC316-BCL-2/Ig H.The recombinant adenoviral plasmid was verified by double digestion and DNA sequencing.Results:(1)Direct sequence analysis on amplified t(14;18) PCR products of BCL-2/Ig H target gene confirmed that the strcture of the j-bcl-2 junctions of the Do HH2 cell line.(2)The BCL-2/Ig H gene was successfully cloned into p UC57-Amp vector, which was further used to carry the exogenous gene into the adenovirus vector.(3)BCL-2/Ig H gene was successfully constructed into the p DC316 of recombinant adenovirus shuttle plasmid type 5, which was identified by sequencing as same as the sequence in Genebank.Conclusion:(1)The shuttle plasmid of r Ad5 carrying human BCL-2/Ig H gene has been successfully constructed and identified, which has laid the foundation for the next step to develop recombinant adenovirus expressing BCL-2/Ig H gene.(2)The shuttle plasmid of r Ad5 carrying human BCL-2/Ig H gene has been successfully constructed and identified, which may be a useful gene transfer tool for the immune target therapy of BCL-2/Ig H positive lymphoma animal mode.(3)The shuttle plasmid of r Ad5 carrying human BCL-2/Ig H gene has been successfully constructed and identified, which may lay the foundation for the research of targeted gene therapy of specific CCND1/Ig H fusion gene of mantle cell lymphoma.