Construction And Expression Of A Prokaryotic Expression Plasmid Of Idiotypic Vaccine Against B Cell Lymphoma: Encoding The Fusion Genes Of Single-chain Variable Fragment And MCP3

Objective:The surface idiotype of immunoglobulin (Ig) expressed on B cell lymphoma cells is a kind of well-known tumor-associated antigen. Injectig the idiotype of Ig combined with a suitable adjuvant to a B cell lymphoma patient can activate patient's immune system and, therefore, eliminate the minimal residual tumor cells by the activated immune system, which may be a novel and practical way to cure lymphoma. The DNA sequence of the single chain variable fragment (scFv) of Ig light chain can be combined with that of Ig heavy chain by gene recombination technology. After being transformed into special cells, the recombined gene can encode and transcriptate a"target"protein, which mimics the antigenicity of integrated Ig. But this"target"protein failed to elicit anti-tumor activity because the weakness of its immunogenicity.The present study is thus designed to clone the scFv gene of the idiotypic Ig from A20, a lymphoma cell line derived from the BABL/c mouse suffering from B cell malignancy, and then to recombine the scFv gene with the gene of monocyte chemotantic protein-3 (MCP3), a suitable adjuvant, and finally, to express the fusion protein vaccine in prokaryotic expressing system for further investigating the effect of idiotype fusion protein vaccine in vivo in animals.Methods:We have used the technique of splicing overlap extension by the polymerase chain reaction to hybrid two different genes together. In this study, we use Splicing by overlap extension by PCR (also recombinate PCR) technique for two times. Firstly, we cloned the cDNAs of immunoglobulin Ig VH and IgVL by RT-PCR and assembled them into the single-chain variable fragment(scFv) by recombinant PCR method. The cDNAs of IgVH and IgVL were connected by a gene fragment encoding a (Gly4Ser) 3 linker. Then, the fragments of scFv and MCP3 were connected with a gene sequence encoding a NDAQAPKS spacer, using recombinant PCR method again. The spacer will ensure the scFv and MCP3 protein folding respectively to maintain their natural function. The fusion gene of scFv-MCP3 were constructed correctly and cloned into the prokaryotic expression plasmid successfully identified by sequencing and restriction endonucleases SacⅠand KpnⅠ. The prokaryotic expression plasmid was transferred to E.coli DH5αto express fusion protein under induction.1 Cells culture and total RNA preparation: A20 cells derived from mouse B cell lymphoma were cultured in DMEM medium with high glucose. Cells were harvested at logarithmic growing phase. Total RNA was extracted using TRIzol Reagent according to the manufacturer's instructions and the integrity of extracted RNA was confirmed by agarose electrophoresis.The amount of RNA was measured by spectrometry.2 Amplification of IgVH,IgVL cDNA fragments by reverse transcription PCR: The total RNA extracted from A20 cells was used as template. Random primers with six nucleotides and the reverse transcriptase of M-MLV were used for reverse transcription. The IgVH and IgVL cDNA fragments were specifically synthesized with primers VH 05'and VH0 3', VL0 5'and VL0 3', respectively. The PCR products were identified by electrophoresis on agarose gel and confirmed by DNA sequencing.3 Amplification of scFv fragment by overlap extension PCR: There are two steps. Firstly, the IgVH and IgVL cDNA fragments were modified by means of PCR using the specific primers VH 5'and VH 3',VL 5'and VL 3', respectively. The ends of the PCR products contain complementary sequences. Secondly, the two modified PCR products were mixed with equal quantity, denatured and reannealed; the single-stranded DNA strands having the complementary sequences annealed and then act as primers for each other. Amplified with primer VH 5'and VL 3'for several circles, the original IgVH and IgVL cDNA were spliced together into scFv.4 Amplification of MCP3 gene: The plasmid encoding MCP3 gene was used as template, primer MCP3 5'and MCP3 3'were applied to amplify MCP3 gene.5 Amplification of scFv-MCP3 fusion fragment by overlap extension PCR: The method of overlap extension PCR was the same as the construction of scFv. Templates of scFv and MCP3 fragments were mixed in equal quantity and primers of VH 5'and MCP3 3'were used to fuse scFv and MCP3 fragment.6 Construction of cloning plasmid encoding scFv-MCP3 fusion gene: scFv-MCP3 fusion gene and T cloning vector were ligated together according to the manufacturer's instructions. The ligatition products were transformed to compentence E.coli DH5 cells. The transformed E.coli DH5 cells were planted onto LB plate,which had been duplicated with antibiotic,X-Gal. The cells were incubated overnight at 37°C. White clonies, which may be the inserts containing colonies, were picked up and macrocultureed. Plasmid DNA was extracted and screened by PCR, restriction endonucleases examination and DNA sequencing with primer T7 and Sp6.7 Construction of protocaryon expressing plasmid encoding scFv-MCP3 fusion gene: The correct clonal plasmid encoding scFv-MCP3 fusion gene and the expressing plasmid pGLo were both cleavaged by restriction endonucleases SacⅠand KpnⅠand the target genes were connected together with T4 ligase. The ligatition products were transformed to compentence E.coli DH5 cells. The transformed E.coli DH5 cells were planted onto LB plate, which had been duplicated with antibiotic. The cells were incubated overnight at 37°C. Several white clonies were picked up and macrocultured. Plasmid DNA was extracted, screened by PCR and restriction endonucleases examination. 8 Expression of scFv-MCP3 fusion protein: E.coli DH5αcells containing recombinant protocaryon expressing plasmid were grown in LB broth at 37°C. When OD600 of the culture became 0.6, L-arabinose was added at a final concentration of 0.01%. The culture was shaked at 30°C for 3.5 h to express target protein. Samples were collected from the culture and the target protein was detected on 10% SDS-PAGE.Results: 1 Amplification of target genes and their identifications by sequencing: The expected size of IgVH and IgVL cDNA fragments amplified by RT-PCR was about 400bp. The sequencing maps indicated: The nucleotide sequence of IgVH cDNA was the same as the data from Genebank. The size of the IgVH, IgVL cDNA and MCP3 modified fragment amplified by overlap extension PCR are 407bp, 401bp and 335bp. The scFv and scFv-MCP3 fragments splicinged by overlap extension were 793bp and 1111bp.The results of sequencing the recombinant cloning plasmid by primer T7 and Sp6 suggested that the scFv-MCP3 fusion gene were connected correctly without frameshift mutation. But there was a silent point mutation happened on the site of 231th nucleotide with A to G changing but the amino acids sequence was not changed. 2 Identification of the recombinant expression plasmid:The recombinant expression plasmid was digested by restriction endonucleases of SacⅠand KpnⅠinto two fragments, 5400bp and 1111bps, respectively. The result suggested that the expression plasmid pGLo/scFv-MCP3 was constructed successfully. 3 Expression of fusion protein: High yield of the fusion protein was expressed. And the fusion protein was 65 kD and account for 30% of the total protein of the bacteria analysised by SDS-PAGE.Conclusion:1 A prokaryotic plasmid, encoding the fusion gene of single-chain variable fragment with MCP3 and expressing idiotypic protein vaccination against B cell lymphoma, was constructed correctly and primarily expressed in DH5α. 2 The mismatching rate of gene splicing by overlap extension PCR is higher than common PCR. So it is necessary to use DNA polymerase of high fidelity to avoid point mutation. 3 We simplified the classic method of gene splicing by overlap extension PCR. Adding the equal-molar ratios'templates to the reaction system at the same time, the sufficient-specific products will be amplified after 30 cycles. 4 BamH I was designed on the origination and termination of MCP3 fragment in the recombinant expression plasmid pGLo/scFv-MCP3. It is convienent to cut MCP3 out to substitution other immune adjuvant.