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Specific anti-human liver cancer phage single-chain antibody HscFv4-16 gene and its uses

A single-chain antibody, phage technology, applied in applications, antibodies, genetic engineering, etc., to achieve strong specificity

Inactive Publication Date: 2008-08-13
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few reports on the screening and identification of anti-hepatoma scFv. So far, there are only small-scale identification reports of anti-hepatoma scFv using liver cancer cell lines and normal human fetal liver cell lines or liver cancer cell lines and other tumor cell lines.
[0003] Use phage display technology to construct an anti-hepatoma phage scFv library, and screen out new anti-hepatoma scFv genes from the library. It is useful in China to identify anti-hepatoma scFv of liver cancer cell lines and normal human fetal liver cell lines or liver cancer cell lines and other tumor cell lines However, there is no report on identifying the specificity of anti-hepatoma scFv using various liver cancer cells and various control cells, human liver cancer tissues and various control tissues

Method used

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  • Specific anti-human liver cancer phage single-chain antibody HscFv4-16 gene and its uses
  • Specific anti-human liver cancer phage single-chain antibody HscFv4-16 gene and its uses
  • Specific anti-human liver cancer phage single-chain antibody HscFv4-16 gene and its uses

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Experimental program
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Embodiment 1

[0039] 1. Acquisition of HscFv 4-16 gene

[0040] 1. Construction of anti-human liver cancer phage single-chain antibody library:

[0041] ① BALB / c mice were immunized by intraperitoneal injection of mixed liver cancer cell line (SMMC-7721 and HepG2) suspension. ②The mouse spleen was removed, and the total RNA of spleen cells was extracted with Trizol reagent, Poly AT tract @ The mRNA purification system purifies total RNA to obtain mRNA. ③Using mRNA as a template, it is reverse transcribed into cDNA. ④ Using cDNA as a template, use the light chain primers of the antibody to amplify the light chain genes (VL) of the complete set of antibodies, and the heavy chain primers of the antibodies to amplify the heavy chain genes (VH) of the complete set of antibodies. ⑤ Recombine the two into a single-chain antibody gene (VL-Linker-VH) using the connecting fragment Linker, that is, the scFv gene. Then, the scFv gene was ligated into pCANTAB 5E vector. ⑥Introduce the recombinant c...

Embodiment 2

[0065] Example 2 Anti-liver cancer experiment of HscFv 4-16

[0066] (1) Experimental method and process

[0067] 1. Mass preparation of target phage antibody HscFv 4-16:

[0068]① Take 100 μL of HB2151 bacteria infected with phage containing the HscFv 4-16 gene, add it to 100 ml of freshly prepared 2×YTAG medium, and shake at 210 rpm at 30°C for 1 hour. ②Centrifuge at 1500g×20min, remove the supernatant. Suspend the precipitate in 100 ml of freshly prepared 2×YTAI medium (without glucose), shake at 210 rpm at 30° C. overnight. ③Centrifuge at 1500g×20min, carefully absorb the supernatant, and filter with a 0.45 micron filter. ④ Precipitate the filtrate with ammonium sulfate. ⑤ Centrifuge at 3000×g for 30 min, discard the supernatant, and suspend the pellet in PBS buffer 1 to 2 times the volume of the pellet. ⑥ Centrifuge to remove residual insoluble matter. ⑦Put the resulting HscFv 4-16 protein solution into a dialysis bag, dialyze to remove ammonium sulfate, and store a...

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Abstract

The invention discloses an astopic anti-hepatocarcinoma phage single-chained antibody HscFv 4-16 gene and appliance, which is characterized by the following: screening gene in the anti-hepatocarcinoma phage single-chained antibody store to select one astopic anti-hepatocarcinoma phage single-chained antibody HscFv 4-16; possessing high astopic reaction of anti-hepatocarcinoma scFv liver cancer cell system and human liver tissue with lowest combination drip rate than normal liver cell system, stomach cancer cell system and colonocarcinoma cell system by 256 times; making positive rate of reacting rate with human liver cancer tissue at 5.0 percent without combination reaction with normal liver tissue, esophagus cancer tissue and lung cancer tissue; reacting positive rate of liver cancer tissue higher than other tissues obviously. The invention possesses strong astopic property, which can be applied in kinds of liver cancer treatment drug and relative agent.

Description

technical field [0001] The invention relates to the field of genetic engineering antibodies, in particular to a specific anti-human hepatocarcinoma phage single-chain antibody (Single chain fragment variable, scFv) HscFv 4-16 gene and application thereof. Background technique [0002] Liver cancer is one of the malignant tumors that seriously endanger human health. People have been looking for various ways to diagnose and treat liver cancer for many years. With the continuous development of tumor immunology and molecular biology technology, there are currently two ways to obtain anti-hepatoma scFv. One is to use genetic engineering technology to transform anti-hepatoma monoclonal antibody into scFv. The transformed anti-hepatoma scFv includes: Hep27 scFv, scFv25, HAb18 scFv, and MDscFv, these scFv have the same specificity as their parental antibodies, their antigen-binding sites have not changed, and their application value is limited by the parental antibodies. The second...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/18C12N15/13C12N15/63A61K39/395A61P35/00
Inventor 杨冬华汤绍辉黄卫舒建昌周旻卢筱华叶刚
Owner JINAN UNIVERSITY
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