ABSTRACT
<p><b>OBJECTIVE</b>To clone the variable region genes of the monoclonal antibody (McAb) against human heterogeneous nuclear ribonucleoprotein A2/B1 (HnRNPA2/B1), ligate them to assemble single chain Fv (ScFv) gene and express in Escherichia coli.</p><p><b>METHODS</b>The specificity of the anti-HnRNPA2/B1 McAb 3E8 to synthetic HnRNPA2/B1 peptide, HnRNPA2/B1 protein in lung cancer cells were examined by dot-immunobinding assay, Western blot and immunohistochemistry. The variable region genes of heavy chain (VH) and light chain (VL) were amplified from hybridoma cell by reverse transcription-polymerase chain reaction(RT-PCR), and then were linked by a linker peptide using SOE-PCR (splicing by overlap extension-PCR) to construct recombination ScFv gene. The latter was cloned into the expression vector pET28 (a+) and expressed in E coli BL21. The expressed product was identified by SDS-PAGE and competitive ELISA inhibition test.</p><p><b>RESULTS</b>It was shown that the McAb combined specifically with synthetic HnRNPA2/B1 peptide and HnRNPA2/B1 protein in three lung cancer cells. The cloned VH gene and VL gene were 345 bp and 309 bp respectively and were linked successfully to obtain ScFv gene. The ScFv protein was expressed in the form of inclusion body, with molecular weight of 28,000 and immunoreactivity to HnRNPA2/B1.</p><p><b>CONCLUSION</b>VH gene, VL gene and ScFv gene of anti-HnRNPA2/B1 antibody were cloned, constructed and functionally expressed in E coli. These results provide the experimental basis for elucidating the role of HnRNPA2/B1 in lung cancer.</p>
Subject(s)
Humans , Adenocarcinoma , Metabolism , Pathology , Antibodies, Monoclonal , Genetics , Allergy and Immunology , Metabolism , Carcinoma, Small Cell , Metabolism , Pathology , Carcinoma, Squamous Cell , Metabolism , Pathology , Cell Line, Tumor , Cloning, Molecular , Escherichia coli , Metabolism , Heterogeneous-Nuclear Ribonucleoprotein Group A-B , Allergy and Immunology , Immunoglobulin Fragments , Genetics , Immunoglobulin Heavy Chains , Genetics , Immunoglobulin Light Chains , Genetics , Immunoglobulin Variable Region , Genetics , Lung Neoplasms , Metabolism , PathologyABSTRACT
<p><b>OBJECTIVE</b>To investigate whether basic fibroblast growth factor (bFGF) can induce the proliferation, invasion and angiogenesis of ovarian cancer or not.</p><p><b>METHODS</b>Human ovarian cancer cell lines SKOV(3) 1 x 10(4)/ml were plated in 24-well dishes, bFGF at 5, 10,15 and 20 ng/ml was added and crystal violet staining was given daily for 8 days, cell numbers were counted by determining OD490. SKOV(3) cells were plated in the center of 50% extra cellular matrix gel, bFGF at 5 and 10 ng/ml was added and the migration distance of cells was measured daily. SKOV(3) 5 x 10(7)/ml were transplanted to BALB/c nude mice subcutaneous. One week later, bFGF, bFGF-MAb or 0.9% nature sodium was injected subcutaneously surrounding the tumor twice a week. Eight weeks later, the experiment ended and the volume of the tumors were measured. Intratumoral microvessel density (MVD) was measured by immunohistochemistry staining for factor VIII.</p><p><b>RESULTS</b>bFGF at 0-10 ng/ml could stimulate the proliferation of SKOV(3) concentration dependently (P<0.05). On the fifth day, the cell proliferation in 10 ng/ml group was 121% above control. bFGF could stimulate the invasion of SKOV(3) concentration dependently (P<0.05). On the seventh day, the migration distance in 5 ng/ml group was 1.16 cm and 153% above control, and that in 10 ng/ml group was 1.86 cm and 245% above control. The average volume of transplanted tumors and MVD in bFGF group were 180% and 146% above control respectively those in bFGF-MAb group were 63.7% and 62.8% above control respectively.</p><p><b>CONCLUSION</b>bFGF can stimulate proliferation, invasion and angiogenesis of ovarian cancer markedly; bFGF-MAb can inhibit the angiogenesis and growth of ovarian cancer.</p>