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Preparation method and application of humanized neutralized anti-influenza NS1 genetic engineering antibody

A genetically engineered antibody and genetic engineering technology, applied in the field of preparation of humanized monoclonal antibodies, can solve the problems of difficult control of influenza, no cross-protection, frequent outbreaks, etc.

Inactive Publication Date: 2010-05-26
HENAN UNIV OF CHINESE MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to its strong antigenic variability and wide host range, there is almost no cross-protection between subtypes, making influenza difficult to control and frequent outbreaks

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1: Antigen Preparation

[0016] Use genetic engineering technology to recombine influenza virus NS1 gene into expression vector pTXB 1 to transform competent E.coliBL21(DE3), pick a single colony on LB(Amp+) agar plate, inoculate in 5ml LB(Amp+) medium, 37℃ Shake the culture overnight, and replant the next day at a ratio of 1:100, expand the culture to A600=0.5-0.7, add a final concentration of 1.0mmol / LIPTG, express at 37°C for 6 hours, and equilibrate the chitin affinity column with 50ml Columnbuffer. Take the supernatant of ultrasonic broken bacteria and load it, use 80ml Column buffer to remove impurity protein, and then use 30ml 30mmol / L DTT [cutting buffer before use (20mmol / LTris-HCl 500mmol / L NaCl, 0.1mmol / L EDTA pH 8.0) Dilution] Rinse the column slowly, close the sample outlet, and cut overnight at 4°C. The next day, rinse with 30ml cutting buffer without DTT, collect the target protein peak, put the obtained target protein solution in a dialysis bag,...

Embodiment 2

[0017] Example 2: Biological screening of phage antibody library

[0018] Coat the 96-well ELISA plate with 150 μl of 100 g / ml NS1 antigen solution (dissolved in 0.1 mol / L NaHCO3 at pH 8.6), and incubate overnight at 4°C. The coating solution was discarded, filled with 1% BSA blocking solution, and reacted at 4°C for 2 hours. Wash the plate 6 times quickly with TBS buffer. Dilute 2×10 with 100 μl of TBST [Tris-HCl buffer + 0.1% (V / V) Tween-20] buffer 11 Pfu of phage was then added to the well-coated wells and shaken gently at room temperature for 60 min. Wash the plate 10 times with TBST buffer, then add 0.2mol / LGlycine-HCl (pH 2.2) to elute for 15min, then use 1mmol / L Tris-HCl 15μl (pH 9.1) to neutralize the above eluate, that is, the first round of specific sex-binding phage. The first round of screening was amplified and purified, followed by the second and third affinity screening. The amount of phage added each time was 2×10 11 pfu, the amount of antigen coated in ...

Embodiment 3

[0019] Example 3: Amplification and purification of specific phage

[0020] Inoculate a single colony of E.coliER2738 in 20ml LB medium, and culture on a shaker until the logarithmic phase. Add unamplified eluate. Incubate vigorously at 37°C for 4.5 hours. Then centrifuge at 10000×g for 10 min at 4°C. Take the supernatant and centrifuge at 10000×g. Transfer the upper 80% of the supernatant to a fresh tube and add 1 / 6 PEG / NaCl. Phage were precipitated overnight at 4°C. Centrifuge at 10,000×g for 15 minutes at 4°C. Discard the supernatant and centrifuge briefly. The pellet was resuspended in 1ml TBS and centrifuged at 4°C for 5min to pellet residual cells. Take the supernatant and reprecipitate with 1 / 6 volume of PEG / NaCl. Incubate on ice for 60min. Centrifuge at 10,000×g at 4°C for 10 min, discard the supernatant. The pellet was resuspended in 200 μl TBS, 0.02% NaN3. Centrifuge at 10,000×g for 1 min, and take the supernatant, which is the eluate after amplification. ...

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PUM

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Abstract

The invention relates to a preparation method and an application of a humanized anti-influenza virus NS1 protein neutralized antibody. Antibodies capable of being combined with the antigenic specificities of influenza A viruses NS1 are directly screened from a human antibody gene bank through applying a gene engineering technology and a phage surface display technology so as to obtain and express antibody genes of the antibodies. The antibody is used for evaluating clinical medicaments for resisting the influenza A viruses and treating the influenza A viruses.

Description

technical field [0001] The invention relates to a preparation method and application of a humanized monoclonal antibody, in particular to a preparation method and application of a humanized anti-influenza virus NS1 protein neutralizing antibody. Background technique [0002] Influenza A is an acute, zoonotic respiratory infectious disease caused by influenza A virus. It is highly contagious and could easily cause a worldwide pandemic. Influenza pandemics have claimed the lives of many people in history. According to the announcement issued by the World Health Organization (WHO), there are 600 million to 1.2 billion influenza cases in the world every year, and 500,000 to 1 million deaths, of which 300 are severe influenza cases. Ten thousand to five million cases, the fatality rate of severe influenza is 8% to 10%. The influenza A(H1N1) epidemic that broke out in Mexico in 2009 and spread rapidly around the world has caused nearly 100,000 infections and thousands of deaths,...

Claims

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Application Information

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IPC IPC(8): C07K16/10G01N33/577A61K39/42A61P31/16
Inventor 杨丽萍袁福宁
Owner HENAN UNIV OF CHINESE MEDICINE
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