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Novel HIV recombined multi-epitope fusion antigen and application thereof

A recombinant antigen and multi-epitope technology, applied in the field of molecular biology and immunology, can solve the problem of clinical value to be evaluated, and achieve the effect of reducing non-specific adsorption, improving efficiency, and not easy to miss detection.

Inactive Publication Date: 2012-07-11
北京万达因生物医学技术有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to avoid infection during the window period, the Netherlands, France and other countries have developed fourth-generation ELISA screening reagents that can simultaneously detect antigens and antibodies, but their clinical value remains to be evaluated

Method used

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  • Novel HIV recombined multi-epitope fusion antigen and application thereof
  • Novel HIV recombined multi-epitope fusion antigen and application thereof
  • Novel HIV recombined multi-epitope fusion antigen and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Application of Recombinant Fusion Protein in HIV ELISA Detection

[0044] 1. Preparation of enzyme-linked plate coated with recombinant antigen: Dilute pTXB1HIVM recombinant antigen with 0.05M, pH9.6 carbonic acid buffer, preferably at a concentration of 2ug / ml, add 100ul / well to the enzyme-linked plate, and keep at 4°C for 24 hours. Discard the liquid, add blocking solution at 200ul / well, and keep at 4°C for 24 hours. Discard the liquid, dry at room temperature less than 30% humidity for 24 hours, and store in vacuum for later use. The blocking solution is 0.02M phosphate buffer containing 0.5% Casein, 10% calf serum, 2% sucrose and 0.1% proclin-300.

[0045] 2. Enzyme conjugate preparation: HRP was labeled with pTXB1HIVM, and the optimal concentration of the enzyme conjugate was selected by conventional square titration method.

[0046] 3. Preparation of quality control serum: collect HIV positive serum, mix more than 5 parts, measure its OD value, finally dilute to...

Embodiment 2

[0058] Application of recombinant fusion protein in the detection of HIV gold standard test strips

[0059] 1 Preparation of coating membrane: 0.02M pH 7.2 phosphate buffer solution is preferred as the coating solution. After filtering through a 0.22um membrane, store it at 4°C for later use, and the validity period is one week. Debug the film spraying machine (Bio-Dot), the preferred film spray volume is 20ul / 30cm, dilute recombinant pTXB1HIVM antigen with coating buffer, the concentration is 0.5mg / ml, dilute the prepared "sheep anti-pTXB1HIVM" with coating buffer "Polyantibody" to a concentration of 0.5mg / ml, machine scribing, the distance between the two lines is 5mm, it should be fine and uniform, and dry at room temperature for 20 minutes. Post-dry at 37°C for 2 hours, seal the bag and prepare the test cardboard for pasting.

[0060] 2 Preparation of the composite pad: 1200ml of deionized water is placed in a clean glass vessel (siliconization is possible if possible), a...

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Abstract

The invention provides a novel HIV recombined multi-epitope fusion antigen and a detection kit for preparing through the antigen. Flexible structured genes are added into the main epitope genes of HIV-1 gp41, M group and O group, the main epitope genes of HIV-1gp120 and the main epitope genes of HIV-2gp36, so that all the gene DNA are linked to be an artificial gene sequence. The sequence is cloned to an expression vector for ectopic expression, so as to establish immunocompetent high-expression cell strain, finally the sequence is fermented and induced into efficient expression, and chromatographic separation and purification is performed on the multi-antigen epitope fusion antigen of the HIV. The antigen is applied to the immune detection reagent, as the unnecessary gene sequence is reduced through the fusion antigen, the non-specific action is reduced for more than 50%, and the synergistic effect of the multi-antigen epitope of the same antigen increases the detection sensitivity to be 2 to 3 times.

Description

technical field [0001] The invention belongs to the fields of molecular biology and immunology, and relates to a recombinant fusion protein, including the vector construction of the DNA sequence encoding the protein, the construction of engineering bacteria and the immunological application of the recombinant fusion protein. Background technique [0002] AIDS is a disease caused by Human Immunodeficiency Virus (HIV). Human immunodeficiency virus was first identified in the United States in 1981. It is a lentivirus that infects cells of the human immune system and is a type of retrovirus. A deadly infectious disease for which there is currently no effective treatment. This virus destroys the immune ability of the human body, causes the immune system to lose resistance, and causes various diseases and cancers to survive in the human body, and develops to the end, leading to AIDS (acquired immunodeficiency syndrome). [0003] At present, 208 countries and regions in the worl...

Claims

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Application Information

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IPC IPC(8): C12N15/62C07K19/00C12N15/63G01N33/68G01N33/571
Inventor 江必胜江洪林昊宇张宝林
Owner 北京万达因生物医学技术有限责任公司
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