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A hepatitis C virus antibody detection reagent comprising recombinant fusion antigen a and b and its application and recombinant fusion antigen a and b

A hepatitis C virus and fusion antigen technology, applied in the field of bioengineering, can solve the problems of poor antigen stability, incomparable reagent performance, incorrect folding, etc., to reduce the risk of sample missed detection, optimize clinical detection effect, The effect of good stability

Active Publication Date: 2020-06-09
深圳德睿生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the preparation of HCV recombinant antigens has technical bottlenecks such as difficult expression or incorrect folding, most of the HCV recombinant antigens independently developed in China are inclusion bodies, with poor activity, and some have poor antigen stability.
The performance of the reagents developed with it can not be compared with the same type of diagnostic reagents abroad, there are many false positives and false negatives, and the problem of low sensitivity
The lack of HCV recombinant antigens with good activity and high stability is the main reason for the inability to prepare domestic detection reagents that meet the requirements of clinical diagnosis

Method used

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  • A hepatitis C virus antibody detection reagent comprising recombinant fusion antigen a and b and its application and recombinant fusion antigen a and b
  • A hepatitis C virus antibody detection reagent comprising recombinant fusion antigen a and b and its application and recombinant fusion antigen a and b
  • A hepatitis C virus antibody detection reagent comprising recombinant fusion antigen a and b and its application and recombinant fusion antigen a and b

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Preparation of recombinant fusion antigen A:

[0039] What this embodiment discloses is the preparation method of recombinant fusion antigen A, specifically as follows:

[0040] 1. Artificially synthesized recombinant fusion antigen A gene sequence and constructed expression vector:

[0041] Sangon Bioengineering (Shanghai) Co., Ltd. artificially synthesized the gene according to the sequence information of the recombinant fusion antigen A gene, and its sequence is SEQ ID NO:1. The gene is linked with the pMD-18 vector after gene synthesis is completed, and the 5' end of the recombinant fusion antigen A gene sequence is designed with a BamH I restriction site, and the 3' end is designed with a terminator and an EcoR I restriction site. The recombinant fusion antigen A gene sequence was double-digested from the pMD-18 vector with BamH I and EcoR I restriction sites, and the pTYB21 vector expression vector was double-digested with BamH I and EcoR I restricti...

Embodiment 2

[0052] Example 2: Preparation of recombinant fusion antigen B:

[0053] This embodiment discloses a preparation method of recombinant fusion antigen B, specifically as follows:

[0054] 1. Artificially synthesized recombinant fusion antigen B gene sequence and constructed expression vector:

[0055] Sangon Bioengineering (Shanghai) Co., Ltd. artificially synthesized the gene according to the sequence information of the recombinant fusion antigen B gene, and its sequence is SEQ ID NO:3. After the gene is synthesized, it is linked to the pMD-18 vector. The 5' end of the recombinant fusion antigen B gene sequence is designed with an EcoR I restriction site, and the 3' end is designed with a terminator and an XhoI restriction site. Use the combination of EcoR I and XhoI restriction sites to double-digest the recombinant fusion antigen B gene sequence from the pMD-18 vector, and use the combination of EcoR I and XhoI restriction sites to double-digest the pET28a(+) expression ve...

Embodiment 3

[0066] Example 3: GST-recombinant fusion antigen B:

[0067] The difference between this example and Example 2 is that this example discloses a GST-recombinant fusion antigen B, which has a GST tag, and the specific preparation method differs from Example 2 only in that:

[0068] The expression vector in step (1) is pGEX-4T-1; the control in step (2) adopts the host bacterium transformed into pGEX-4T-1 empty vector; in step (3), add GST Loadingbuffer to replace His Loading buffer, GST ElutionBuffer to replace His Elution Buffer, Glutathione Sepharose 4Fast Flow affinity column replaces NiSepharose TM 6Fast Flow affinity column, PBS buffer replaces Tris buffer.

[0069]Among them, the composition of GST Loading buffer is: NaCl 8.18g / L, KCl 0.2g / L, NaCl 2 HPO 4 3.58g / L, KH 2 PO 4 0.25g / L, pH7.3. The composition of GST Elution Buffer is: Tris 6.06g / L, reduced glutathione 3.04g / L, pH8.0. The composition of PBS buffer is: Na 2 HPO 4 5.8g / L, NaH 2 PO 4 0.6g / L, NaCl 9.0g...

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Abstract

The invention relates to an HCV (hepatitis c virus) antibody detection reagent containing recombinant fusion antigens A and B, an application and the recombinant fusion antigens A and B, and belongs to the technical field of bioengineering. According to the HCV antibody detection reagent containing the recombinant fusion antigens A and B, the recombinant fusion antigen A contains an amino acid sequence as shown in SEQ ID NO: 2; the recombinant fusion antigen B contains an amino acid sequence as shown in SEQ ID NO: 4; the detection reagent comprises a reagent R1, a reagent R2, a reagent R3 anda reagent R4. The detection reagent has the following advantages: the two recombinant fusion antigens have the characteristics of high specificity and good stability and can be used for preparing thehepatitis C virus antibody detecting reagent by mixing in a certain ratio, and the clinical detection effect of the reagent is remarkable. Meanwhile, the detection reagent has high anti-interference capacity to chylomicron, bilirubin and hemoglobin, and can greatly meet requirements of clinical diagnosis of HCV.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a hepatitis C virus antibody detection reagent comprising recombinant fusion antigens A and B and its application and recombinant fusion antigens A and B; in particular, it relates to a method using different hepatitis C virus fusion antigens Fragments are combined to prepare recombinant fusion antigen A and recombinant fusion antigen B. At the same time, the two recombinant fusion antigens are applied to the preparation of hepatitis C virus antibody detection reagents and the application of clinical detection. Background technique [0002] Hepatitis C is an infectious disease that is caused by hepatitis C virus (HCV) infection and seriously threatens human health. It is mainly transmitted by blood and body fluids. According to World Health Organization estimates, there are approximately 130-170 million hepatitis C virus-infected people worldwide. The anti-HCV positive rat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N33/569C12N15/62C07K19/00
CPCC07K14/005C07K2319/00C12N2770/24222G01N33/56983G01N33/6854G01N2333/186G01N2469/20
Inventor 周珂
Owner 深圳德睿生物科技有限公司
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