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Gene engineering bacterium for preparing Ebola virus nucleoprotein antigen and application

A technology of genetically engineered bacteria and Ebola virus, applied in the direction of viruses/bacteriophages, viruses, viral peptides, etc., can solve the problems of difficult large-scale preparation, low protein expression, low sensitivity, etc., and achieve simple, fast, and remarkable specificity in the preparation process Non-degradable, non-degradable effect

Pending Publication Date: 2018-07-31
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the specificity of ELISA detection methods established with different target antigens is quite different. The research by Groen and Sobarzo et al. showed that the sensitivity of ELISA detection methods established with Ebola virus VP35, VP40 and GP as antigens was lower than that of ELISA detection results using Ebola nucleoprotein NP as antigen (Groen et al., 2003; Sobarzo et al., 2012)
Moreover, in the above-mentioned studies on the preparation of antigens using classical prokaryotic expression systems and eukaryotic expression systems (including baculovirus expression systems and mammalian cell expression systems), although good detection sensitivity and specificity have been achieved, there are still protein expressions. The disadvantages such as small amount, tedious virus culture process, and easy cell contamination make most of the preparation methods, especially the purification process, complex and cumbersome, difficult to quickly prepare large quantities, and the quality of antigens is not easy to control. For example, purified antigens are prone to precipitation, Thereby greatly reducing its use efficiency and effect as an indirect ELISA detection antigen
In addition, the cost of antigen preparation in eukaryotic expression systems is usually high, and these factors are undoubtedly obstacles to the convenient and widespread use of Ebola virus antibody indirect ELISA detection

Method used

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  • Gene engineering bacterium for preparing Ebola virus nucleoprotein antigen and application
  • Gene engineering bacterium for preparing Ebola virus nucleoprotein antigen and application
  • Gene engineering bacterium for preparing Ebola virus nucleoprotein antigen and application

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Experimental program
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Effect test

Embodiment 1

[0039] A genetically engineered bacteria for preparing Ebola virus nucleoprotein antigen is obtained by the following steps:

[0040] A. PCR-amplified gene: the original gene template name pCRII-enp, provided by the research group of Wei Hongping, Wuhan Institute of Virology, Chinese Academy of Sciences (verified by sequencing). The forward primer is 5ˊ-GGATCCATGGATTCTCGTCCTCAGAAAAT-3ˊ(F), and the reverse primer is 5'-GTCGACTCACTGATGATGTTGCAGGATT-3'(R). The PCR reaction reagents are as follows: 5μL of 10×ExTaq polymerase buffer, 1μL of 2.5mM dNTP Mixture, 1 μL of primer F (10uM), 1 μL of primer R (10uM), 1 μL of amplification template, 1 μL of ExTaq HS DNA polymerase (5U / ul) and 40 μL of sterile water, PCR reaction conditions: 94°C Pre-denaturation for 300 seconds; denaturation at 94°C for 30 seconds, renaturation at 58°C for 45 seconds, extension at 72°C for 30 seconds, 35 cycles; final extension at 72°C for 600 seconds. The PCR amplification product was subjected to agarose...

Embodiment 2

[0044] The preparation method of Ebola virus nucleoprotein antigen comprises the following steps:

[0045] A. Inoculate the cloned strain pET-28a-ZEBOV-NP / BL21(DE3) into 5 mL of fresh LB medium (add 50 mg / ml kanamycin at 1:1000), and cultivate overnight.

[0046] B. Take an appropriate amount of culture and add it to fresh LB at 1:100 (at the same time, add 50mg / ml kanamycin at 1:1000 to inhibit the growth of miscellaneous bacteria), incubate at 37°C for 3 hours, and measure OD600=0.2-0.4.

[0047] C. Add the inducer IPTG to induce protein expression. The induction conditions are: IPTG concentration 0.4mM, induction temperature 37°C, induction time 3h. After induction, the cells were isolated by centrifugation at 8000 rpm for 10 min.

[0048]D. Pour out the medium residue, resuspend the cells with an appropriate amount of PBS (usually use 1 / 10 volume of the culture liquid), ultrasonically disrupt the cells to OD600<0.1, centrifuge at 12,000 rpm for 10 minutes, and separate th...

Embodiment 3

[0051] Western-blot detection of Ebola virus nucleoprotein, the steps are:

[0052] A. Ebola virus nucleoprotein prepared by the above method and corresponding negative control and positive control are subjected to SDS-PAGE electrophoresis ( image 3 );

[0053] B, 100mA, 1 hour, transfer the sample to PDVF membrane;

[0054] C. 5% (m / v) skimmed milk powder, blocked overnight at 4°C, and washed 3 times with TBST (containing 0.05% (v / v) Tween-20), with an interval of 10 minutes each;

[0055] D. Dilute the self-prepared specific polyclonal antibodies (Ebola virus nucleoprotein antibody ENP and Marburg virus nucleoprotein antibody MNP) to 1:2000 times as the primary antibody, and incubate at room temperature for 1.5 hours;

[0056] E. After washing 3 times with the same method, add 2000-fold diluted secondary antibody-AP-labeled goat anti-rabbit IgG (Beyotime, CAT NO: A0239), and incubate at room temperature for 1.5 hours;

[0057] F. After washing, the membrane was added to ...

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Abstract

The invention discloses a gene engineering bacterium for preparing an Ebola virus nucleoprotein antigen and application. A gene engineering strain Escherichia coli Bl21(DE3) / pET-28a-EBOV-NP is inducedto express Ebola virus nucleoprotein, and the antigen is obtained through centrifugation and ultrasonic crushing. The antigen is strong in solubility, has good antigenicity and is suitable for serving as an antigen for indirect ELISA detection. The preparation method of the Ebola virus nucleoprotein antigen is simple and rapid, and the yield is large. Compared with a classical preparation method,the obtained antigen is more stable in property, and an established indirect ELISA detection method is high in sensitivity, good in specificity, short in detection period and wide in application range.

Description

technical field [0001] The invention belongs to the technical field of pathogen detection, and in particular relates to a genetically engineered bacterium for preparing Ebola virus nucleoprotein antigen and its application. Using the engineering bacterium provided by the invention, the Ebola virus nucleoprotein antigen can be rapidly prepared in large quantities. It can be specifically combined with the corresponding antibody, the antigen has strong sensitivity and remarkable specificity in the detection of Ebola virus nucleoprotein antibody, and has the application value of being developed into a detection kit. Background technique [0002] Ebola virus (EBOV) can cause Ebola hemorrhagic fever. The main symptoms are fever, diarrhea, hemorrhage inside and outside the body, etc. It is mainly transmitted through contact with body fluids and mucous membranes, and aerosol transmission. The rate is extremely high, and there are no approved therapeutic drugs and vaccines, so it is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P21/02G01N33/569C12R1/19
CPCG01N33/56983C12P21/02C07K14/005C12N2760/14122C12N2760/14131
Inventor 黄弋袁志明朱友杰杨梦诗
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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