Construction method of expression vector for preparing membrane protein CD14 antibody

A technology for expressing vectors and construction methods, applied in biochemical equipment and methods, vectors, nucleic acid vectors, etc., can solve the problems of poor recognition of membrane proteins and low expression of CD14 antibodies, and achieve good recognition and high-efficiency expression effects

Inactive Publication Date: 2017-05-31
WUHAN GENECREATE BIOLOGICAL ENG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Aiming at the problems existing in the prior art, the object of the present invention is to provide a method for constructing an expression vector for preparing the membrane protein CD14 antibody. The expression vector cons

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  • Construction method of expression vector for preparing membrane protein CD14 antibody
  • Construction method of expression vector for preparing membrane protein CD14 antibody
  • Construction method of expression vector for preparing membrane protein CD14 antibody

Examples

Experimental program
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Effect test

Embodiment 1

[0022] A method for constructing an expression vector for preparing membrane protein CD14 antibody of the present invention comprises the following steps:

[0023] Step (1): Immunize the mice with human peripheral blood mononuclear cells (PBMC) as the immunogen, boost the immunization once 3 days before taking the mouse spleen, measure the titer of antiserum, take the mouse with high titer, extract Spleen total RNA, design specific primers at both ends of the sequence, the primer sequence is shown in the nucleotide sequence list, respectively use the primer VH pair and the primer VL pair to amplify the variable region of the heavy chain and the variable region of the light chain; the amplification reaction conditions are as follows : 94°C pre-denaturation for 5min, 94°C for 45s, 58°C for 1min, 72°C for 45s, a total of 30 cycles, 72°C extension for 10min;

[0024] Step (2): Link the gene of the helper expression sequence to the upstream of the heavy chain variable region by SOE...

Embodiment 2

[0030] The method for preparing the membrane protein CD14 antibody through the expression vector constructed in Example 1 is as follows: mix the obtained pFUSEss_CHIg heavy chain expression plasmid and pFUSE2ss_CLIg light chain expression plasmid plasmid, and transfect them into 293T cells through PEI sustained release solution. 293T cells were subcultured to ensure that the confluence of the cells reached 80% to 85%. Mix the DNA dilution and PEI dilution, let it stand for 5 minutes, add it to the 293T cells, culture it in a carbon dioxide incubator at 37°C, and take a sample for detection after 48 hours of transfection Whether the antibody is expressed or not, the sampling time is determined according to the detection situation; the cell line 293T cells containing the two plasmids are screened with resistance, and the CD14 antibody is obtained after the cell line is cultured.

[0031] 1. Detection of 293T cell line by ELISA

[0032] Coat the ELISA plate with anti-human KAPPA ...

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Abstract

The invention discloses a construction method of an expression vector for preparing a membrane protein CD14 antibody. The method comprises the following steps of immunizing mice by adopting a peripheral blood mononuclear cell (PBMC) as an immunogen, extracting spleen total RNA (Ribose Nucleic Acid), and sequentially designing specific primers on two ends; respectively connecting an expression sequence-assisting gene, a histidine-tag gene and a beta2 microglobulin gene to a heavy chain variable region and a light chain variable region so as to obtain a target fragment; respectively introducing two enzyme sites to an upstream of the heavy chain variable region and a downstream of the light chain variable region, cloning the target fragment onto a carrier pFUSEss-CHIg-hG1 through a PCR technology, and acquiring the expression vector for preparing the membrane protein CD14 antibody. The expression vector constructed by the method can carry out high-efficiency expression so as to solve the problem of low expression quantity of the CD14 antibody.

Description

technical field [0001] The invention relates to the technical field of expression vectors, in particular to a method for constructing an expression vector for preparing membrane protein CD14 antibody. Background technique [0002] There are two types of CD14, mCD14 and sCD14, which are glycoproteins on the cell surface. Play an important role in pathological reactions such as shock. Screening out antibodies that can recognize CD14 molecules plays an important role in the study of the function of CD14 molecules and the entire pathway. If the function of CD14 can be completely blocked, and the combination of CD14 and LPS and LPS / LBP complex can be specifically blocked, the occurrence of pathological reactions such as LPS inflammatory response and endotoxin shock can be prevented or stopped, which is very important for clinical treatment of endotoxemia. , Endotoxin shock, etc. will also have better application prospects. [0003] The cost of membrane protein expression is re...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/66C12N15/65
CPCC12N15/65C12N15/66C12N15/85C12N2800/107
Inventor 华权高沈鹤霄马峰罗绍祥肖颖舒芹徐春雷王静邓陈玲易汪雪李昆鹏
Owner WUHAN GENECREATE BIOLOGICAL ENG CO LTD
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