| Acute leukemia remains the major life-threatening disease in children. At present, combined chemotherapy with multiple anti-cancer agents is still the main modality of treatment for this disease. Although most chemotherapies are working effectively on low risk patients, severe side effects and poor long-term prognosis are the two major expected obstacles for the successful treatment of this common pediatric malignancy due to the poor treatment selection of the chemotherapeutic agents. As compared to conventional chemotherapy, monoclonal antibody (McAb) targeting therapy is more effective with less side effects owing to its perfect selectivity and specificity. Unfortunately, most McAbs currently available with high affinity are mouse origin. The clinical application of these mouse antibodies in patients has been greatly restricted due to the occurrence of severe serum sickness and human anti-mouse immunoglobulin antibody (HAMA) in patients during treatment. So it is necessary to reduce the immunogenicity of the mouse antibody to human body maximally and keep their antigen recognizing activity. As we all know, the immunogenicity of a given antibody mainly exists in its constant (Fc) region while the antigen binding activity is located atits variable (Fv) region. In order to make the mouse antibodies eligible for clinical use, modification of antibody molecule is urgently required. One of the practical methods is to reconstruct the single chain fragment of variable region (ScFv) gene and expression of its antibody protein using modern genetic engineering techniques.B lineage acute lymphoblastic leukemia (ALL) is the common type of hematopoietic malignancies in children. Immunophenotyping analysis of leukemia cells have showed that CD 19, an antigen consistently and steadily expressed on almost all differentiation stages of B lineage cells, thus this particular antigen might be an ideal target for antibody-targeting treatment of B lineage malignancy.At present, many kinds of anti-human CD 19 McAbs are being studied in the world. Most results have demonstrated that both conjugated and unconjugated McAbs were the efficient targeting agents to B lineage ALL cells in either animal model or clinical patients. Also, there are evidences to show that not every clone of CD 19 McAbs is working the same and each has its own reactive activity and selective cell kill of the B lineage malignancies. So, it is necessary to reconstruct and study more CD19 clones.There are only two hybridoma cell lines secreting anti-human CD 19 McAbs available in China. One (clone name: CD19.1, IgG1) is in the Hematology Institute of Chinese Academy of Medical Sciences and no report about its reconstruction of this clone has been found in literature. Another clone, ZCH (Zhejiang Children's Hospital)-4-2E8 or simply 2E8 belonging to murine IgM subtype was generated in our laboratory which was assigned to CD 19 category by the 6th International Workshop and Conference on Human Leukocyte Differentiation Antigens (HLDA6) in 1996.Our previous work has successfully cloned the variable region (V) genes of both heavy (H) chain (VH2E8) and light (L) chain (VL2E8) of 2E8 antibody and the sequences were almost accurate. Analyses both online and by DNASIS ver2.5 software have showed that the sequences obtained are within the open reading frames (ORF) of the variable region genes of mouse immunoglobulin. However, the correct sequences of the variable region genes encoding our target antibody 2E8 remain to be confirmed. In this study, our work will be focusing on the cloning and sequencing of the recombinant ScFv2e8 genes, the constructing of the eukaryotic expression vector pSectag2A/ScFv2E8,the establishing of the transgenic Chinese hamster ovary (CHO) cells and the expression of the ScFv2E8 protein and its characterization with a series of molecular biology techniques. Hopefully, the results of this study could provide a fundamental basis for the further engineering of this important antibody with potential of clinical targeting treatment.Material and Methods1. Cell culture: The cell lines were cultured under routine conditions.2. Cloning and sequencing of the VH^es, VL2es and ScFv2es genes: 1 x 107 fresh 2E8 hybridoma cells were harvested by centrifugation to isolate the total RNA using TRIZOL. The VH2E8, VL2E8 and ScFv2es genes were amplified by reverse transcriptase polymerase chain reaction (RT-PCR) and spliced by overlap extension (SOE) methods with specific enzymatic location primer pairs. The sequences were analyzed online by cloning the genes into pGEM?-T easy vectors.3. Construction of ScFv2es eukaryotic expression vector: The ScFv2E8 gene was cloned from pGEM?-T easy vector and reconstructed into the secretive eukaryotic expression vector pSectag2A by using a series enzyme treatments. Positive recombinants were named pSectag2A/ScFv2E8-4. Establishment of the transgenic CHO cell line: pSectag2A/ScFv2E8 was stably transfected into CHO cells by using Superfect transfection reagent. After the screening culture with Zeosin, the transgenic CHO cell line named CHO/pSectag2A/ScFv2E8 was successfully established.4.1. Identification of ScFv2es gene in the transgenic CHO cells: 1 x 10 fresh CHO/ pSectag2A/ScFv2E8 cells were harvested by centrifugation to isolate the total RNA. The" VH2E8, VL2E8 and ScFv2E8 genes were amplified by RT-PCR with specific primer pairs ofPl/P2,P3/P4andPl/P4.4.2. Identification of ScFv2es antibody protein:4.2.1 The ScFv2E8 protein and its molecular weight were analyzed by SDS-PAGE and Western Blot techniques.4.2.2 The ScFv2es protein in the CHO/pSectag2A/ScFv2E8 cell culture supernatant wasevaluated by flow cytometry (FCM): 5 x 105/tube of fresh Naml-6 cells was prepared. lOOul of the transgenic CHO culture supernatant was added into each tube. After 30 min of incubation, the cells were washed twice with PBS by centrifugation. Then 2 ul each of either anti-mouse IgM-FITC or k-FITC was added into each tube with 30 min incubation and washed twice as described above. Then the samples were analyzed by FCM to prove whether the ScFv2E8 protein existed in the supernatant.4.2.3 The ScFv2E8 protein on the CHO/pSectag2A/ScFv2E8 cell membrane was detected by flow cytometry (FCM): Anti-mouse IgM-FITC or k-FITC was added to each tube containing 5 x 105/tube of fresh transgenic CHO cells. After 30 min of incubation, the cells were washed twice with PBS by centrifugation as above. Then FCM analysis was applied to detect the ScFv2E8 protein on the cell membrane.4.2.4 The ScFv2E8 protein in the cytoplasm of CHO/pSectag2A/ScFv2E8 cells was evaluated by flow cytometry (FCM): 5 x 10 /tube of fresh transgenic CHO cells were fixed with 200|til of 2% paraformaldehyde solution for 30min followed by two washes with PBS by centrifugation. The cell membranes were permealized by adding 200ul of permealizing solution into each tube and incubated for another 15 min. After wash twice, 2 ul of either anti-mouse IgM-FITC or k-FITC was added and reacted for 30 min. Again FCM analysis was used to detect whether the ScFv2E8 protein existed inside the cells.4.3 The activity of the ScFv2es protein was evaluated with blocking test: 5 x 105/tube of fresh Naml-6 cells were prepared. One tube as test was added with lOOul of the transgenic CHO culture supernatant and the other tube as negative control was added with the same volume of regular medium. After 30 min of reaction, the cells were washed twice with PBS. Then both tubes were added with appropriate volumes of 2E8-FITC and reacted for 30min followed by two washes. FCM analysis was utilized to observe whether the ScFv2E8 protein activity could be detected in the supernatant.Results1. PCR amplification, TA cloning and sequencing of VH2E8 and VL2E8 genes: The VH2E8 gene of about 400 bps was amplified with #29 and #34 primers designed formurine VH gene while the VL2E8 gene was amplified with #37 and #44 primers designed for murine VL gene. No corresponding bands were amplified from the hybridoma partner — murine myeloma cell line NS-1 with the same pairs of primers. The target genes were cloned and sequenced. The analyses online and by software DNASIS ver2.5 showed that the sequences obtained were within the open reading frames of the mouse immunoglobulin variable region genes, almost accurate to the results obtained by our previous work. Only 4 point mutations were identified and corrected in VH2E8 gene while the VL2E8 sequence was exactly the same as our previous result.2. Cloning and sequencing ofScFv2E8gene: VH2E8 primers P1/P2 and VL2E8 primers P3/P4 were designed and synthesized according to the construction of expression vector and the identification of ScFv2e8 protein. ScFv2E8 gene amplified through splicing by overlap extension (SOE) was cloned into pGEM?-T easy vector. The sequence analysis online and by software DNASIS ver2.5 showed that it contained start codon ATG, IgK-chain leader sequence, ORF, 6 x His and stop codon TGA.3. Construction of ScFv2es secreting eukaryotic expression vector: ScFv2E8 gene segment and pSectag2A vector segment treated with Sfil and EcoRI enzymes were ligated together by T4 DNA ligase. The sequence analysis online and by software DNASIS ver2.5 showed that it contained the inserted gene with start codon ATG, IgK-chain leader sequence, ORF, 6 x His and stop codon TGA.4. Expression and identification of ScFv2es protein: pSectag2A/ScFv2E8 was stably transfected into CHO cells after culture screening with Zeosin. A transgenic CHO cell line named CHO/pSectag2A/ScFv2E8 was successfully established.4.1 Identification of ScFv2esgsne inside the transgenic CHO cells: Fresh CHO cells were harvested by centrifugation to isolate the total RNA. The bands indicating the VH2E8, VL2e8 and ScFv2es genes were clearly shown on the gel after RT-PCR amplification of CHO/pSectag2A/ScFv2E8 RNA with corresponding pairs of primers as P1/P2, P3/P4 and P1/P4, respectively while those bands were not found on the negative control RNA template derived from CHO/pSectag2A cells. These results indicated that there exists the ScFv2E8 mRNA in the transgenic CHOcells. The transfection was successful.4.2 SDS-PAGE analysis of ScFv2E8protein: The CHO/pSectag2A/ScFv2E8 cell culture supernatant was analyzed by SDS-PAGE. The results showed that a great deal of nonspecific protein strips were observed and no target protein strip could be identified.4.3 Western Blot analysis: The CHO/pSectag2A/ScFv2E8 cell culture supernatant was analyzed by Western Blot with the anti-6> |
PDF Full Text Request