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Cloning of Anti-Human Epidermal Growth Factor Receptor 2 Nanobody Gene by pComb3xss Vector into ER2738 Bacterial System
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| Author:
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RANA SH ISMAIL, AWATIF H ISSA, ABDULELAH A ALMAYAH, MAHMOUD GANJI
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| Abstract:
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Antibody-mediated therapy for cancer treatment relies on the identification of selected molecular targets. This study is aimed to cloning monoclonal nanobodies into ER2738 (host bacterial cell), this antibody is derived from immunized camel with the purified recombinant HER2 protein. Following camel’s immune response was measured by ELISA which showed the high titter (1/3200). Lymphocytes from the camel’s blood were isolated, then total RNA was extracted, and first strand of cDNA was synthesized. The heavy chain antibody was amplified by nested PCR using (FPF-1 and FPRCH2) specific primers. The second PCR run was to amplify VHH gene (which is encoded for nanobodies) by using (VHH-F-PRIMER and MMA-VHH FOR 2) specific primers. Both of plasmid pComb3XSS and PCR production (VHH) was digested using sfiI enzyme (restriction enzyme) and both of them was ligated using T4DNA ligase enzyme and then transferred into E.coli ER2738 (host bacterial cell). The result of transformation efficiency showed (9 × 106 cfu/ml). The transformation was confirmed by PCR with specific primers.
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| Keyword:
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Nanobody, Cloning, pComb3XSS.
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| EOI:
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-
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| DOI:
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https://doi.org/10.31838/ijpr/2021.13.01.729
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| Download:
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Request For Article
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