Lentivirus expression vector of anti-p185erdB2 human mouse chimeric antibody containing F/2A sequence and construction method of lentivirus expression vector

A p185erbb2, human-mouse chimeric antibody technology, applied in botanical equipment and methods, biochemical equipment and methods, introduction of foreign genetic material using vectors, etc. Expression imbalance, etc.

Inactive Publication Date: 2012-08-01
广西壮族自治区肿瘤防治研究所
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Problems solved by technology

However, these methods have some defects, such as low initiation efficiency of expressed genes, mutual interference between promoters, unbalanced expression of upstream genes and downstream genes, etc., resulting in significant differences in the expression levels of light and heavy chains, resulting in complete antibody low molecular expression

Method used

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  • Lentivirus expression vector of anti-p185erdB2 human mouse chimeric antibody containing F/2A sequence and construction method of lentivirus expression vector
  • Lentivirus expression vector of anti-p185erdB2 human mouse chimeric antibody containing F/2A sequence and construction method of lentivirus expression vector
  • Lentivirus expression vector of anti-p185erdB2 human mouse chimeric antibody containing F/2A sequence and construction method of lentivirus expression vector

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Embodiment Construction

[0032] Anti-p185 with F / 2A sequence erbB2 Human-mouse chimeric antibody lentiviral expression vector and its construction method

[0033] 1 Materials and methods

[0034] 1.1 Plasmids, strains and cells

[0035] The three-plasmid lentivirus system consists of expression plasmid pWPI, structural plasmid pCMV-dR 8.74 and envelope plasmid pMD2G, donated by the Laboratory of Clinical Oncology Center, University of Ottawa, Canada. The plasmid pUC57 / F2A containing the F / 2A sequence was constructed by Shanghai Sangon Company. Transformed with Escherichia coli DH-5α containing anti-p185 erbB2 Plasmid pWPI / H-IRES-L and 293T cells of human and mouse chimeric light and heavy chain genes are preserved by Guangxi Zhuang Autonomous Region Institute of Cancer Prevention and Control.

[0036] 1.2 Tool enzymes and main reagents

[0037] Restriction enzymes BamH I, Pvu II, Nsi I, Xba I, Pme I, T4 DNA ligase, etc. were purchased from New England Biotechnology Company. Taq DNA polymerase, m...

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Abstract

The invention discloses a lentivirus expression vector of an anti-p185erdB2 human mouse chimeric antibody containing an F/2A sequence, and a construction method of the lentivirus expression vector. Furin or foot and mouth disease virus/2A polypeptide (F/2A) with self shearing capacity is connected with a heavy chain and a light chain of the human-mouse chimeric antibody to form an open reading frame (ORF), and the ORF is inserted into a lentivirus expression vector pWPI to construct the expression vector pWPI/H-F2A-L of the recombination anti-p185erdB2 overall-length human mouse chimeric antibody. According to sequencing identification, the pWPI/H-F2A-L is consistent to expected design. The infection efficiency of recombination lentivirus is 87.68%, and the expressions of the chimeric heaving chain and light chain are formed after the recombination lentivirus is infected with 293T cells. Compared with that of a lentivirus expression vector of an IRES (Internal Ribosome Entry Site) chimeric antibody, the expression level of the chimeric antibody mediated by the F/2A is higher than that of the chimeric antibody mediated by IRES, thereby laying a foundation for research on anti-p185erdB2 engineering antibodies.

Description

technical field [0001] The invention relates to a lentiviral expression vector, especially an anti-p185 containing F / 2A sequence erbB2 Human-mouse chimeric antibody lentiviral expression vector and its construction method. Background technique [0002] c-erbB2 / HER2 / neu encoded product p185 erbB2 It is a transmembrane glycoprotein with tyrosine kinase activity and belongs to the epidermal growth factor receptor (EGFR) family. Amplification of c-erbB2 / HER-2 / neu gene and p185 erbB2 Overexpression of the protein is seen in a variety of malignancies, including breast, ovarian, colon, endometrial, gastric, prostate, and lung adenocarcinomas. p185 erbB2 Overexpression indicates poor prognosis of tumors, such as metastasis, recurrence, easy drug resistance and short survival period. p185 erbB2 As a tumor antigen, it is an ideal target for tumor therapy. The U.S. Food and Drug Administration (FDA) officially approved the introduction of an anti-p185 strain in October 1998. er...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/66
Inventor 李力刘芳张玮
Owner 广西壮族自治区肿瘤防治研究所
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