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HIV-2 recombinant antigen and its preparation method and application

A HIV-2 and recombinant antigen technology, applied in the field of HIV detection, can solve the problems of limited effect and high price, and achieve the effects of increasing the frequency of repeated use, improving specificity, and good freeze-thaw stability

Active Publication Date: 2021-11-09
SICHUAN ANKERUI NEW MATERIAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The direction of prevention and treatment of AIDS is mainly three aspects of vaccine, diagnosis and treatment. Among them, the development of HIV vaccine has not been successful so far; the treatment of HIV is not only expensive, but also has extremely limited effect; basic, most effective

Method used

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  • HIV-2 recombinant antigen and its preparation method and application
  • HIV-2 recombinant antigen and its preparation method and application
  • HIV-2 recombinant antigen and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Design and Construction of expression vectors Example 1 Recombinant gp36 Antigen

[0077] Using genetic engineering techniques, to be the direction of the dominant epitope region antigen research, the inventors have designed a large number of HIV-2gp36 antigenic fragments, wherein only the gp36-1 (SEQ ID NO.1), gp36-2 (SEQ ID NO .2), gp36-3 (SEQ ID NO.3) and gp36-4 (SEQ ID NO.4) By way of example, the experimental procedure described screening.

[0078] Select 553-668aa region of the full-length HIV-2 antigen, named gp36-1, the method of codon optimized chemical synthesis of nucleotide sequences obtained gp36-1 (SEQIDNO.5); HIV-2 antigen selected Full 538-668aa long region, named gp36-2, the codon-optimized chemical synthesis method to obtain a nucleotide sequence gp36-2 (SEQIDNO.6); 523-668aa selected region of the full-length HIV-2 antigen named gp36-3, the codon-optimized chemical synthesis method to obtain a nucleotide sequence gp36-3 (SEQIDNO.7); and selecting region 5...

Embodiment 2

[0084] Example 2 Rear Grouping GP36 antigen induced expression and purification

[0085] The positive recombinant plasmid of sequencing, transformed E. coli E.Colibl21 (Rossett) in Escherichia coli was converted to LB agar medium containing 100 μg / ml AMP + and 25 μg / ml CHL +. Under 37 ° C, inverted culture overnight or incubation of 12 to 14 h to long out of single colonies.

[0086] Two single bacteria were collocated to 5 ml of LB liquid medium containing 100 μg / ml amp + and 25 μg / ml CHL +, and 200 rpm was cultured overnight. The next day, 200 μl of bacteria was transferred to 5 mL of fresh lt of LB liquid medium containing 100 μg / ml amp + and 25 μg / mlCHL +, 200 rpm was cultured to OD600 about 0.8. Take 1 ml of bacterial solution as a sample before induction, and IPTG was added to the remaining bacteria to a final concentration of 1 mm, and the oscillation culture was continued at 37 ° C for 4 to 5 h. SDS-PAGE electrophoresis after the end of the induction (see Figur...

Embodiment 3

[0090] Example 3 recombinant GP36 antigen protein replication

[0091] The eluate was collected, loaded into a 3000-3500 DA molecular weight dialysis bag, and complement the destination protein at 2 ~ 8 ° C:

[0092] Buffera: 10mm CB, 0.05% SDS, 10% glycerol, 1mm DTT, 4M urea, pH 10.2;

[0093] Bufferb: 10mm Cb, 0.05% SDS, pH10.2.

[0094] First use buffera (1L) dialysis purposes, from about 2 to 4 h, the equivalent Bufferb (2L) is added to Buffera, and then dialyzes about 2 to 4 h. Thereafter, 1 L of the dialysate containing 2 m urea was poured, and 1 l bufferb was added thereto, and the dialysis was continued for 2 to 4 h. Finally, it is transferred to a new 2L BufferB dialysis, dialysis 3 times, each dialyzing 2 to 4h. After the dialysis is completed, the protein is removed from the dialysis bag.

[0095] The reproached target protein was concentrated with ultrafiltration, then centrifuged at 12000 rpm at 2-8 ° C for 20 min, collected in 15 ml of centrifuge tube, and the antige...

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Abstract

The present invention relates to HIV‑2 recombinant antigen, the amino acid sequence of which is shown in SEQ ID NO.1 or the derived amino acid sequence obtained by substitution, deletion or addition of one or several amino acids from the amino acid sequence shown in SEQ ID NO.1 , the derived amino acid sequence has the activity of the amino acid sequence shown in SEQ ID NO.1. The recombinant protein of the present invention can be used for HIV detection, and has improved performance in the development of in vitro diagnostic kits. The present invention also relates to corresponding polynucleotides, constructs, host cells, fusion proteins, compositions, kits and preparation methods.

Description

Technical field [0001] The present invention relates to the detection of HIV, particularly to HIV-2 recombinant antigens for immunoassays. Background technique [0002] AIDS (acquired immune system deficiency, AIDS) is a viral disease threatening human health the world's attention. The main pathogen that causes AIDS is the type I and type II human immunodeficiency virus (HIV-1 and HIV-2). HIV infection due to immune defect levels deeper, from asymptomatic carriers to develop severe immune failure, leading to the development of a variety of fatal diseases and death. According to WHO statistics, by the end of 2018 the global HIV-infected persons is estimated at 37.9 million cases, only the 2018 global new HIV infections by up to 1.7 million cases. In developing countries, due to various reasons such as economy, culture and education, HIV prevalence intensified, some countries have lost control of the epidemic, while my country has also become the early stages of the outbreak of the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/155C12N15/49C12N15/70C12N1/21C07K19/00G01N33/569G01N33/68
CPCC07K14/005C07K2319/00C12N15/70C12N2740/16222G01N33/56983G01N33/6854G01N2333/161G01N2469/20
Inventor 曾红蔡泉蜀干盈盈何涛
Owner SICHUAN ANKERUI NEW MATERIAL TECH CO LTD
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