Bifunctional antibody, construction method thereof, and bifunctional antibody gene engineering drug

A technology of bifunctional antibody and construction method, which is applied in the field of biopharmaceuticals and achieves the effect of broad application prospects

Inactive Publication Date: 2015-02-11
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The technical problem that the present invention hopes to solve is to provide an easy-to-implement method for the construction of a bifunctional an...

Method used

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  • Bifunctional antibody, construction method thereof, and bifunctional antibody gene engineering drug
  • Bifunctional antibody, construction method thereof, and bifunctional antibody gene engineering drug
  • Bifunctional antibody, construction method thereof, and bifunctional antibody gene engineering drug

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Obtaining a stably expressed cell line with the eukaryotic expression vector pBudce4.1

[0051] Fresh human blood was taken, total RNA was extracted with RNA extraction kit, and cDNA was reverse transcribed. The FC gene was amplified with primer 1 and primer 2, the CL gene was amplified with primer 3 and primer 4, the CD4 gene was amplified with primer 5 and primer 6, and the CCR5 gene was amplified with primer 7 and primer 8, respectively. DNA fragments with sizes of 1100bp, 300bp, 600bp and 400bp were obtained after DNA electrophoresis analysis.

[0052] A 1400bp FC-CD4 fragment was amplified with primer 1 and primer 4, FC gene and CD4 gene as templates, and Hind III and BamH I restriction sites were introduced into the fragment respectively. The 700bp CL-CCR5 fragment was amplified with primer 5 and primer 8, CL gene and CCR5 gene as template, and Not I and Kpn I restriction sites were introduced into the fragment respectively.

[0053] The FC-CD4 gene f...

Embodiment 2

[0057] Example 2: A cell line stably expressed with the eukaryotic expression vector pCDNA5 / FRT / TO TOPO TA

[0058] The downstream primer 9 sequence was designed as: 5'-TAG AAG GCA CAG TCG AGG-3'. The expression cassette containing FC-CD4 fragment and expressing CL-CCR5 protein was amplified from the expression plasmid in Example 1 with primer 1 and primer 9. The expression cassette contains the EF-1α promoter sequence, and its size is about 4600bp after DNA electrophoresis analysis. After recovering by DNA gel electrophoresis, take an appropriate amount of PCR products and the carrier pCDNA5 / FRT / TO TOPO TA in proportion to connect with ligase, react at 16°C for 2 hours, transform into E. On the plate, cultivate overnight at 37°C, pick a single clone colony and culture it in LB medium containing ammonium antibiotics. When the OD value of the bacterial solution reached 1.0, the plasmid was extracted to obtain the recombinant plasmid pCDNA5 / FRT / TO TOPO TA-FC-CD4+CL-CCR5 double...

Embodiment 3

[0060] Example 3: Obtain a stably expressed cell line with the eukaryotic expression vector pEGFP-N1

[0061] The sequence of upstream primer 10 was designed as: 5'-CTC CAG GAT AGT GGC ACC TGG TGAGCC CCT CTC CCT CCC CCC CCC-3',

[0062] The downstream primer 11 sequence was designed as: 5'-TGT GGC CAT ATT ATC ATC GTG GACAGA TGG TGC AGC CAC AGT-3',

[0063] The IRES gene (about 500bp) was amplified from the plasmid pIRES2-EGFP with primer 10 and primer 11, respectively, and then the FC-CD4 fragment and the IRES gene were amplified with primer 1 and primer 11 to obtain a FC- CD4-IRES fragment. The IRES-CL-CCR5 fragment of about 1500bp was amplified with primer 10 and primer 8, and the IRES fragment and CL-CCR5 fragment as templates, respectively. The FC-CD4-IRES-CL-CCR5 gene fragment was amplified with primer 1 and primer 8 respectively, using FC-CD4-IRES fragment and IRES-CL-CCR5 fragment as templates, and a gene fragment of about 2900 bp was obtained.

[0064] The FC-CD4-IR...

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Abstract

The invention provides a bifunctional antibody and a construction method thereof. The bifunctional antibody comprises an antibody heavy chain constant area, an antibody light chain constant area, a first target protein, which is connected to the antibody heavy chain constant area, and a second target protein, which is connected to the antibody light chain constant area. The construction method of the bifunctional antibody comprises the following steps: individually obtaining the antibody heavy chain constant area gene, antibody light chain constant area gene, the first target gene, and the second target gene; adopting expression carriers to construct recombinant carrier shuttle plasmids of the genes mentioned above; transfecting the recombinant carrier shuttle plasmids to an expression bacterium strain to carry out culture so as to obtain the bifunctional antibody; carrying out centrifugation, and collecting the supernate or purifying the cells so as to obtain the bifunctional antibody. The construction method assembles antibody molecules in a gene level, thus the pertinence is strong, and the method is easy to achieve. The obtain bifunctional antibody comprises two antigen combining sites, so the bifunctional antibody can combine two different antigens at the same time.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals and relates to a recombinant antibody. More specifically, the present invention relates to a bifunctional antibody comprising two target proteins, its construction method, and a genetic engineering drug containing the bifunctional antibody. Background technique [0002] Naturally occurring antibody molecules have a similar structure, that is, they all have a symmetrical structure composed of two identical heavy chains and two identical light chains. Based on this symmetrical structure, recombinant antibodies similar to natural antibody structures can be designed, that is, the conserved constant region structures of the heavy and light chains are retained, and the variable regions are replaced with molecules of the target antibody. At this time, two different genes linked to the variable region of the heavy chain and the variable region of the light chain not only have the specificity o...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/66C07K19/00
Inventor 易俊波周兆平苏庆宁买制刚章刚王玉树雷明军林枫
Owner SHENZHEN UNIV
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