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HIV-1 integrase expression bacterial strain and integrase inhibitor in-vitro screening model

An HIV-1 and integrase technology, applied in the field of genetic engineering, can solve the problems of unstable experimental results, environmental hazards, and high costs, and achieve the effects of simple and easy operation, low cost, and few interference factors for screening models.

Active Publication Date: 2010-03-17
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Both methods have many disadvantages and are not widely used
The radioactive elements used in the autoradiography method have certain hazards to the experiment operators and are easy to cause pollution to the environment; the improved ELISA method has higher requirements on the operation skills of the experimenters, the experimental results are unstable, the reaction background, and the false positive rate high and costly

Method used

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  • HIV-1 integrase expression bacterial strain and integrase inhibitor in-vitro screening model
  • HIV-1 integrase expression bacterial strain and integrase inhibitor in-vitro screening model
  • HIV-1 integrase expression bacterial strain and integrase inhibitor in-vitro screening model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Embodiment 1: The specific construction steps of the screening model provided by the present invention are as follows:

[0073] 1. Primer synthesis

[0074] Primers were designed according to the integrase gene sequence (from plasmid pNL4-3, available in NCBI):

[0075] His-Primer1: 5'-CAGCATATGTTTTTAGATGGAATAGATAAGGCCCAAGATGAAC-3'

[0076] NdeI restriction site: CATATG; start codon: ATG

[0077] Primer2: 5'-ATAGGATCCTTACTAATCCTCATCCTGTCTACTTGCCACAGAATCATCACCTGCC-3'

[0078] BamHI restriction site: GGATCC, stop codon: TTA, CTA; mutation site: AGA (to obtain C280S);

[0079] Primer3: 5'-CCACAATAAAAAAAGAAAAGGGGGGATTGGG-3'

[0080] Mutation site: AAA (get F185K);

[0081] Primer4: 5'-CCCAATCCCCCCTTTTTCTTTTTTTATTGTGG-3'

[0082] Mutation site: TTT;

[0083] LTR-Primer1: 5'-CAGTGGATCCGACTGTGGAAGGGCTAATTTGGTC-3'

[0084] BamHI restriction site: GGATCC; integrase action site: ACTG;

[0085] LTR-Primer2: 5'-CGACGAATTCGACTGCTAGAGATTTCCAC-3'

[0086] EcoRI restriction s...

Embodiment 2

[0111] Example 2: Construction of an in vitro screening model for HIV-1 integrase inhibitors

[0112] 1. Take out the constructed expression strain BL21-ET28a-IN stored in a glycerol tube from -80°C, inoculate it in 20ml of LB medium containing kanamycin sulfate 60μg / ml at a ratio of 1:1000, at 37°C, 180 rpm, overnight culture;

[0113] 2. The seed bacteria cultivated overnight in the previous step were inoculated into 10 bottles of 200ml LB medium according to 1:100, added kanamycin sulfate to a final concentration of 60μg / ml, 37°C, 180 rpm, and cultivated for 3 hours;

[0114] 3. Add the inducer IPTG to each bottle of culture in step 2 to a final concentration of 0.4mM, 37°C, 150 rpm, and induce expression for 3 hours;

[0115]4. Centrifuge each bottle of culture in step 3 at 5000rpm for 30 minutes to collect the bacteria, dissolve the bacteria in 100ml of 0.01M PBS buffer, freeze at -20°C, and then thaw;

[0116] 5. Ultrasonicate each bottle of culture in step 4 at 300W f...

Embodiment 3

[0120] Example 3: Application of HIV-1 integrase inhibitor in vitro screening model

[0121] 1. Add 20 pmol of the integrase obtained in Example 1, 200 ng pUC19-HIVLTR, 2 mM MnCl, 5 mM β-mercaptoethanol and different concentrations of samples to be tested in 10 μl of 20 mM Tris-HCl reaction system;

[0122] 2. After incubating the reaction system in step 1 at 37°C for 30 minutes, add 1 μl of 10×loading buffer to terminate the reaction, and run 1% agarose gel electrophoresis at 60V for 20 minutes;

[0123] 3. Use the gel imaging system UVP Biolmaging System to observe the plasmid bands in the 2-step electrophoresis, and use LabWorks45Image Acquisition and Analysis Software 4.5.00.0 to analyze the IOD values ​​of supercoiled, linear and open-circular DNA. The results are shown in Table 2. Using this model to detect, the integrase inhibitor standard can show the activity of integrase inhibition, and the result shows that the construction of the model is successful. Using this m...

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Abstract

The invention relates to an HIV-1 integrase expression bacterial strain and an integrase inhibitor in-vitro screening model. In the model, HIV-1 integrase soluble expression plasmid and expression plasmid with integrase LTR sequence are firstly established based on gene engineering technology, and established recombinant plasmid pET28a-IN is transferred into commercial colibacillus BL21 to obtainthe integrase expression bacterial strain; and then the integrase expression bacterial strain is cultured in large-scale, finally integrase is separated and purified, works on the substrate plasmid and is added into samples to measure the activity. The detection model overcomes the defects such as high cost, high background and more influence factors in the existing ELISA detection method; the detection model can carry out primary screening on various natural or chemically synthetic integrase inhibitors in-vitro largely and quickly; and the detection model is an HIV-1 integrase in-vitro screening model with the advantages of quick operation, flexibility, fewer interference factors and low cost.

Description

【Technical field】: [0001] The invention belongs to the technical field of genetic engineering. The invention uses genetic engineering to construct a soluble integrase expression vector and a plasmid with an integrase substrate LTR sequence, uses purified integrase to act on the substrate plasmid, and adds a sample to determine the influence of the sample on the integrase activity. This detection model overcomes the shortcomings of the existing ELISA detection method, such as high cost, high background, and many influencing factors, and can perform preliminary screening of various natural or chemically synthesized integrase inhibitors in vitro in large quantities and quickly. 【Background technique】: [0002] Human acquired immunodeficiency syndrome (AIDS, also known as AIDS) is caused by human immunodeficiency virus (human immunodeficiency virus, HIV). AIDS is a disease with a high infection rate, and the number of HIV infections and deaths worldwide remains high. There is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12Q1/25C12R1/19
Inventor 刘方江筠王常荣李宁陈芝惠张聃吕鹏云曲丽媛乔文涛
Owner NANKAI UNIV
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