CN114354926A - Detection method of echovirus 9 type antibody - Google Patents

Abstract

The invention discloses an echovirus 9-type antibody detection method, which takes ECHO9VP1 recombinant protein as an antigen of an enzyme label plate, breaks through the limitation that the prior enzyme label plate is directly coated by virus strain inactivation as the antigen or the cell is infected by the virus strain to prepare an antigen tablet, and realizes the requirements of high flux and safety in the preparation of the enzyme label antigen plate. The invention is not only beneficial to the clinical treatment of aseptic encephalitis disease, but also plays an important role in the research of early discovery, early intervention and epidemiology of epidemic outbreak caused by ECHO9 and the technical support for guaranteeing the health of people.

Description

Detection method of echovirus 9 type antibody

Technical Field

The invention relates to the technical field of medical treatment, in particular to an echovirus 9 type antibody detection method.

Background

Aseptic encephalitis outbreaks caused by Echovirus type 9 (ECHO 9) have been reported in the united states, canada, uk, europe, japan, korea, and so on. The incidence of viral encephalitis is high in China, but few viral encephalitis caused by ECHO9 are reported, which may be related to the fact that no effective method for detecting ECHO9 exists. At present, the ECHO9 etiology detection mainly adopts virus culture, serum neutralization test or RT-PCR amplification target gene strip, then gene sequencing is carried out, and Blast comparison is carried out on GeneBank website to determine the type of the ECHO9 virus. The methods have the common defects of time and labor waste, incapability of high-throughput detection, higher requirements on laboratory personnel, instruments and equipment, higher cost and incapability of being carried out in a basic laboratory. Therefore, the establishment of the ECHO9 antibody detection method is not only beneficial to the clinical treatment of aseptic encephalitis disease, but also plays a vital role in the early discovery and early intervention of outbreak epidemic situation and the guarantee of the health of people.

Disclosure of Invention

The invention aims to provide a method for detecting an echovirus type 9 antibody. The invention has the advantages of rapid detection and high accuracy, and can meet the requirements of high throughput and safety in detection.

The technical scheme of the invention is as follows: the detection method of the echovirus 9 type antibody comprises the following steps:

s1: preparation of ECHO9 pET28a-VP1 recombinant plasmid: carrying out rare codon optimization of escherichia coli on the ECHO9VP1 sequence, and re-synthesizing the ECHO9VP1 sequence;

plasmid pET28a + expression reading frame design VP1 gene amplification primers:

an upstream primer: 5'-CATATGGGTAGGGTTGCTGACACAATACG-3', containing NdeI restriction site and initiation codon ATG;

a downstream primer: 5'-CTCGAGTTACACATACACAGCTCCAGATTCTTGG-3', containing XhoI cleavage sites;

carrying out double enzyme digestion on the resynthesized ECHO9VP1 sequence and the plasmid pET28a + prokaryotic expression vector by NdeI and XhoI respectively to obtain an ECHO9 pET28a-VP1 recombinant plasmid;

s2: high-efficiency expression of ECHO9VP1 recombinant protein and verification: the recombinant bacterium induced by IPTG is adopted to efficiently express the ECHO9VP1 recombinant protein, and 6 histidines contained at the N tail end of the protein are utilized to be purified and identified by a nickel column affinity chromatography method, so as to obtain the 37kD ECHO9VP1 recombinant protein;

s3: preparing enzyme-labeled antigen plate by preparing 10 microgram/ml recombinant protein solution with pH9.6 and 0.01mol/L carbonate buffer solution, adding 0.1ml to each hole of 96-hole enzyme-labeled plate, and coating overnight at 4 ℃; washing with 0.05% Tween 20-0.01mol/L PBS for 3 times, sealing with 20% calf serum, and sealing the prepared enzyme-labeled antigen plate in a refrigerator at 4 deg.C;

s4 establishment of the detection method of the ECHO9 IgM/IgG antibody in human serum: 1:20 diluted human serum is used as a primary antibody; 1, using HRP-marked goat anti-human IgM diluted by 5000 or using HRP-marked goat anti-human IgG as a secondary antibody and TMB as a chromogenic substrate, establishing detection ECHO9VP 1-IgM/IgG-ELISAs, and determining the OD value of each hole by using a Bio-Rad enzyme labeling instrument after terminating the reaction: the measurement wavelength is 450nm, and the reference wavelength is 630 nm; if the OD light absorption value of the detected specimen is larger than or equal to the negative control mean value +3SD, the specimen is positive.

In the detection method of the echovirus 9-type antibody, in step S1, T4 ligase is adopted for connection, after the connection, a product is transferred into E.coli DH 5 alpha for culture, and a monoclonal is selected for PCR, enzyme digestion and sequencing identification of recombinant plasmids.

In the detection method of the echovirus type 9 antibody, in step S4, the operation steps for detecting the ECHO9 IgM or IgG antibody in human serum are as follows:

s4.1, setting a negative control 2 hole and a positive control 1 hole in a test, and adding 100 mu L of corresponding liquid into each hole; setting blank contrast 1 hole empty;

s4.2, adding physiological saline or PBS 1:100 mu L of 20 diluted serum sample, vibrating, uniformly mixing, sticking a sealing film, and placing in an incubator at 37 ℃ for 30 minutes;

s4.3, preparing 0.05% Tween 20-0.01mol/L PBS washing liquid for later use;

s4.4, placing the plate washing machine to wash the plate, and repeatedly washing the plate for 5 times;

s4.5, adding an enzyme-labeled working solution, wherein each hole is 100 mu L, and a blank hole is not added, and placing the mixture in an incubator at 37 ℃ for 30 minutes;

s4.6, placing the plate washing machine to wash the plate, and repeatedly washing the plate for 5 times;

s4.7, sucking 100 mu L of TMB into each hole, fully mixing the TMB and the hole uniformly, and placing the mixture at room temperature for developing for 10 minutes;

s4.8, immediately adding 100 mu L of stop solution into each hole, and placing the mixture into an enzyme-linked immunosorbent assay device to determine the wavelength to be 450nm and the reference wavelength to be 630 nm. Adjusting zero by using a blank hole, and reading the OD value of each hole; if the OD light absorption value of the detected specimen is larger than or equal to the negative control mean value +3SD, the specimen is positive.

In the foregoing method for detecting an echovirus type 9 antibody, the determining the result of the operation of detecting an ECHO9 IgM or IgG antibody in human serum includes:

if IgM and IgG antibodies in human serum are negative, ECHO9 is not infected;

ECHO9 IgM positive and IgG negative in human serum, which is ECHO9 infection acute phase;

ECHO9 positive IgM and IgG in human serum is ECHO9 infection recovery period;

ECHO9 IgM negative and IgG positive in human serum are ECHO9 past infection.

Compared with the prior art, the enzyme-labeled plate uses the ECHO9VP1 recombinant protein as the antigen of the enzyme-labeled plate, breaks through the limitation that the prior enzyme-labeled plate is directly coated with the virus strain through inactivation of the virus strain or the virus strain infects cells to prepare the antigen tablet, and meets the requirements of high flux and safety in preparation of the enzyme-labeled antigen plate. The invention coats the expressed and purified ECHO9VP1 recombinant protein on an ELISA plate, optimizes ELISA test conditions, and establishes a method for detecting IgM and IgG in human serum against ECHO9 by an indirect ELISA method. The invention is not only beneficial to the clinical treatment of aseptic encephalitis disease, but also plays a vital role in the early discovery and early intervention of epidemic situation and the guarantee of the health of people.

Drawings

FIG. 1 is a flow chart of the present invention;

FIG. 2 is a schematic diagram of the construction of recombinant plasmid ECHO9 pET28a-VP 1;

FIG. 3 is a schematic diagram showing the restriction enzyme identification of the recombinant plasmid pET28a-VP 1;

FIG. 4 is a diagram showing the identification of purified VP1 protein by Western blot;

FIG. 5 is a schematic diagram of Western blot for identifying VP1 protein rabbit antibody;

FIG. 6 is a diagram illustrating the ELISA method for identifying the rabbit antibody titer of VP1 protein.

Detailed Description

The invention is further illustrated by the following figures and examples, which are not to be construed as limiting the invention.

Example (b): an echovirus 9 type antibody detection method is shown in figure 1 and comprises the following steps:

s1: as shown in FIG. 2, ECHO9 pET28a-VP1 recombinant plasmid was prepared: carrying out rare codon optimization of escherichia coli on an ECHO9VP1 sequence published in GenBank, and re-synthesizing an ECHO9VP1 sequence;

plasmid pET28a + expression reading frame design VP1 gene amplification primers:

an upstream primer: 5'-CATATGGGTAGGGTTGCTGACACAATACG-3', containing NdeI restriction site and initiation codon ATG;

a downstream primer: 5'-CTCGAGTTACACATACACAGCTCCAGATTCTTGG-3', containing XhoI cleavage sites;

the resynthesized ECHO9VP1 sequence and plasmid pET28a + prokaryotic expression vector were double digested with NdeI and XhoI, respectively. The DNA fragment is ligated by using T4 ligase, the ligated product is transferred into E.coli DH 5 alpha for culture, and a single clone is picked for PCR as shown in FIG. 3 (note: 1: DNA marker; 2: NdeI and XhoI double digestion pET28a-VP1), and the recombinant plasmid is identified by digestion and sequencing and is named as pET28a-VP 1.

S2: high-efficiency expression of ECHO9VP1 recombinant protein and verification: the recombinant protein ECHO9VP1 is efficiently expressed by recombinant bacteria induced by IPTG, and the recombinant protein ECHO9VP1 is obtained by purifying and identifying 6 histidines contained at the N-terminal of the protein by a nickel column affinity chromatography, as shown in figure 4 (note 1: protein marker; 2: purified VP1 protein). The polyclonal antibody prepared by immunizing a rabbit with the purified VP1 recombinant protein shows that the polyclonal antibody can specifically bind to VP1 protein, as shown in FIG. 5 (note: 1: protein marker antibody; 2: VP1 protein rabbit antibody). The experimental result of FIG. 5 shows that the purified recombinant protein has better antigenicity and can be used as an indirect ELISA diagnostic antigen.

TABLE 1

The ELISA method for identifying the rabbit antibody titer of the VP1 protein is shown in Table 1 and FIG. 6. The purified VP1 recombinant protein 100 ng/well is coated on an enzyme label plate; rabbit serum polyclonal antibody diluted by 1:1000, 1:5000, 1:10000, 1:50000 and 1:100000 is taken as a first antibody; goat anti-rabbit IgG (H + L) as secondary antibody, and 5% PBS milk as primary antibody for negative control; the positive control used anti-His antibody 6E25ug/ml as the primary antibody. Experiments show that the rabbit serum polyclonal antibody can still be combined with the purified VP1 recombinant protein for color development after being diluted by one hundred thousand times. The result shows that the recombinant VP1 protein has good immunogenicity. Wherein from left to right in FIG. 6, the diluted rabbit serum is 1:1000, 1:5000, 1:10000, 1:50000, 1:100000 in 1-5 wells; 6 wells are negative controls.

S3: preparing enzyme-labeled antigen plate by preparing 10 microgram/ml recombinant protein solution with pH9.6 and 0.01mol/L carbonate buffer solution, adding 0.1ml to each hole of 96-hole enzyme-labeled plate, and coating overnight at 4 ℃; washing with 0.05% Tween 20-0.01mol/L PBS (pH7.4) for 3 times, sealing with 20% calf serum, sealing the prepared enzyme-labeled antigen plate in a refrigerator at 4 deg.C, and storing;

s4 establishment of the detection method of the ECHO9 IgM/IgG antibody in human serum: 1:20 diluted human serum is used as a primary antibody; 1, using HRP-marked goat anti-human IgM diluted by 5000 or using HRP-marked goat anti-human IgG as a secondary antibody and TMB as a chromogenic substrate, establishing detection ECHO9VP 1-IgM/IgG-ELISAs, and determining the OD value of each hole by using a Bio-Rad enzyme labeling instrument after terminating the reaction: the measurement wavelength is 450nm, and the reference wavelength is 630 nm; if the OD light absorption value of the detected specimen is larger than or equal to the negative control mean value +3SD, the specimen is positive.

The procedures for detecting ECHO9 IgM or IgG antibody in human serum are as follows:

s4.1, setting a negative control 2 hole and a positive control 1 hole in a test, and adding 100 mu L of corresponding liquid into each hole; setting blank contrast 1 hole empty;

s4.2, adding physiological saline or PBS 1:100 mu L of 20 diluted serum sample, vibrating, uniformly mixing, sticking a sealing film, and placing in an incubator at 37 ℃ for 30 minutes;

s4.3, preparing 0.05% Tween 20-0.01mol/L PBS (pH7.4) washing solution for later use;

s4.4, placing the plate washing machine to wash the plate, and repeatedly washing the plate for 5 times;

s4.5, adding enzyme-labeled working solution (HRP-labeled goat anti-human IgM or IgG), adding 100 mu L of enzyme-labeled working solution into each hole, not adding blank holes, and placing the holes in an incubator at 37 ℃ for 30 minutes;

s4.6, placing the plate washing machine to wash the plate, and repeatedly washing the plate for 5 times;

s4.7, sucking 100 mu L of TMB into each hole, fully mixing the TMB and the hole uniformly, and placing the mixture at room temperature for developing for 10 minutes;

s4.8, immediately adding 100 mu L of stop solution into each hole, and placing the mixture into an enzyme-linked immunosorbent assay device to determine the wavelength to be 450nm and the reference wavelength to be 630 nm. Adjusting zero by using a blank hole, and reading the OD value of each hole; if the OD light absorption value of the detected specimen is larger than or equal to the negative control mean value +3SD, the specimen is positive.

The judgment of the result of the detection operation includes:

if IgM and IgG antibodies in human serum are negative, ECHO9 is not infected;

ECHO9 IgM positive and IgG negative in human serum, which is ECHO9 infection acute phase;

ECHO9 positive IgM and IgG in human serum is ECHO9 infection recovery period;

ECHO9 IgM negative and IgG positive in human serum are ECHO9 past infection.

The invention coats the expressed and purified ECHO9VP1 recombinant protein on an ELISA plate, optimizes ELISA test conditions, and establishes a method for detecting IgM and IgG in human serum against ECHO9 by an indirect ELISA method. The results of the ECHO-9 epidemic positive serum detected by the ELISAs method established by the invention are consistent with those of the German Severe echovirus detection kit. By using the ELISA method established by the invention, 176 parts of serum of febrile children under the less than fourteen years old with negative rubella and measles IgM antibody detection in Hangzhou areas and 240 parts of serum of physical examination children are screened by ECHO 9. And comparing the result with a German Severe Exxon virus detection kit, and finding that the detection positive rate of the method is close to that of the German Severe kit. The present invention also found that the antibody positivity rates for febrile children were comparable with the two methods, but the antibody ECHO9 positivity rates for healthy children were more than doubled. After comparison of the two methods, no statistical difference was found for the febrile children, 2 ═ 1.647 and P ═ 0.199. But for healthy children, 2 ═ 5.47, P ═ 0.019; the two methods are statistically different.

In conclusion, the ECHO9VP1 recombinant protein is used as the antigen of the enzyme label plate, the limitation that the prior enzyme label plate is directly coated with the virus strain through inactivation of the virus strain or the virus strain infects cells to prepare the antigen tablet is broken through, and the requirements of high flux and safety in preparation of the enzyme label plate are met. The invention is not only beneficial to the clinical treatment of aseptic encephalitis disease, but also plays an important role in the research of early discovery, early intervention and epidemiology of epidemic outbreak caused by ECHO9 and the technical support for guaranteeing the health of people.

Claims (4)

1. A method for detecting an echovirus type 9 antibody, which is characterized by comprising the following steps: the method comprises the following steps:

s1: preparation of ECHO9 pET28a-VP1 recombinant plasmid: carrying out rare codon optimization of escherichia coli on the ECHO9VP1 sequence, and re-synthesizing the ECHO9VP1 sequence;

plasmid pET28a + expression reading frame design VP1 gene amplification primers:

an upstream primer: 5'-CATATGGGTAGGGTTGCTGACACAATACG-3', containing NdeI restriction site and initiation codon ATG;

a downstream primer: 5'-CTCGAGTTACACATACACAGCTCCAGATTCTTGG-3', containing XhoI cleavage sites;

carrying out double enzyme digestion on the resynthesized ECHO9VP1 sequence and the plasmid pET28a + prokaryotic expression vector by NdeI and XhoI respectively to obtain an ECHO9 pET28a-VP1 recombinant plasmid;

s2: high-efficiency expression of ECHO9VP1 recombinant protein and verification: the ECHO9VP1 recombinant protein is efficiently expressed by recombinant bacteria induced by IPTG, and the protein is purified and identified by a nickel column affinity chromatography method by utilizing 6 histidines contained at the N tail end of the protein, so as to obtain the 37kD ECHO9VP1 recombinant protein;

s3: preparing enzyme-labeled antigen plate by preparing 10 microgram/ml recombinant protein solution with pH9.6 and 0.01mol/L carbonate buffer solution, adding 0.1ml to each hole of 96-hole enzyme-labeled plate, and coating overnight at 4 ℃; washing with 0.05% Tween 20-0.01mol/L PBS for 3 times, sealing with 20% calf serum, and sealing the prepared enzyme-labeled antigen plate in a refrigerator at 4 deg.C;

s4 establishment of the detection method of the ECHO9 IgM/IgG antibody in human serum: 1:20 diluted human serum is used as a primary antibody; 1, using HRP-marked goat anti-human IgM diluted by 5000 or using HRP-marked goat anti-human IgG as a secondary antibody and TMB as a chromogenic substrate, establishing detection ECHO9VP 1-IgM/IgG-ELISAs, and determining the OD value of each hole by using a Bio-Rad enzyme labeling instrument after terminating the reaction: the measurement wavelength is 450nm, and the reference wavelength is 630 nm; if the OD light absorption value of the detected specimen is larger than or equal to the negative control mean value +3SD, the specimen is positive.

2. The method for detecting an echovirus type 9 antibody according to claim 1, wherein: in step S1, T4 ligase is used for ligation, the product is transferred to e.coli DH 5 α for culture, and a single clone is selected for PCR, enzyme digestion and sequencing to identify the recombinant plasmid.

3. The method for detecting an echovirus type 9 antibody according to claim 1, wherein: in step S4, the procedures for detecting ECHO9 IgM or IgG antibodies in human serum are as follows:

s4.1, setting a negative control 2 hole and a positive control 1 hole in a test, and adding 100 mu L of corresponding liquid into each hole; setting blank contrast 1 hole empty;

s4.2, adding physiological saline or PBS 1:100 mu L of 20 diluted serum sample, vibrating, uniformly mixing, sticking a sealing film, and placing in an incubator at 37 ℃ for 30 minutes;

s4.3, preparing 0.05% Tween 20-0.01mol/L PBS washing liquid for later use;

s4.4, placing the plate washing machine to wash the plate, and repeatedly washing the plate for 5 times;

s4.5, adding an enzyme-labeled working solution, wherein each hole is 100 mu L, and a blank hole is not added, and placing the mixture in an incubator at 37 ℃ for 30 minutes;

s4.6, placing the plate washing machine to wash the plate, and repeatedly washing the plate for 5 times;

s4.7, sucking 100 mu L of TMB into each hole, fully mixing the TMB and the hole uniformly, and placing the mixture at room temperature for developing for 10 minutes;

s4.8, immediately adding 100 mu L of stop solution into each hole, and placing the mixture into an enzyme-linked immunosorbent assay device to determine the wavelength to be 450nm and the reference wavelength to be 630 nm. Adjusting zero by using a blank hole, and reading the OD value of each hole; if the OD light absorption value of the detected specimen is larger than or equal to the negative control mean value +3SD, the specimen is positive.

4. The method for detecting an echovirus type 9 antibody according to claim 3, wherein: the judgment of the result of the ECHO9 IgM or IgG antibody detection operation in human serum comprises the following steps:

if IgM and IgG antibodies in human serum are negative, ECHO9 is not infected;

ECHO9 IgM positive and IgG negative in human serum, which is ECHO9 infection acute phase;

ECHO9 positive IgM and IgG in human serum is ECHO9 infection recovery period;

ECHO9 IgM negative and IgG positive in human serum are ECHO9 past infection.

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