阅读说明：本技术 一种SARS-CoV-2冠状病毒IgA抗体及其在ELISA检测试剂盒中的应用 (SARS-CoV-2 coronavirus IgA antibody and its application in ELISA detection kit ) 是由 戴方平 陈东生 程艳兵 张韦唯 于 2020-12-25 设计创作，主要内容包括：本发明公开一种SARS-CoV-2冠状病毒IgA抗体,一种SARS-CoV-2冠状病毒IgA抗体及其在ELISA检测试剂盒中的应用,包括下列技术措施：1)根据SARS-CoV-2冠状病毒侵入人体后自身产生的所有抗体全长序列信息与未感染SARS-CoV-2冠状病毒者体内抗体序列进行比对分析；2)并进行了原核密码子优化,并将pET32a—IgA质粒转化至E.coli BL21(DE3)感受态中,在E.coliBL21(DE3)菌株中高效表达并纯化,最后获得纯化的IgA蛋白。3)鉴定人群是否感染SARS-CoV-2冠状病毒或者检测人血清中是否含有SARS-CoV-2冠状。具有灵敏度高,特异性好以及纯度高等特点,本发明的ELISA试剂盒灵敏度较高,特异性较强,操作简单,检测时间短,临床实用性强。(The invention discloses a SARS-CoV-2 coronavirus IgA antibody, a SARS-CoV-2 coronavirus IgA antibody and its application in ELISA detection kit, comprising the following technical measures: 1) comparing and analyzing the full-length sequence information of all antibodies generated by SARS-CoV-2 coronavirus after invading human body with the in-vivo antibody sequence of the patient without SARS-CoV-2 coronavirus infection; 2) and prokaryotic codon optimization is carried out, pET32a-IgA plasmid is transformed into E.coli BL21(DE3) competence, and is efficiently expressed and purified in E.coli BL21(DE3) strain, and finally, purified IgA protein is obtained. 3) Identifying whether the human population is infected with SARS-CoV-2 coronavirus or detecting whether the human serum contains SARS-CoV-2 coronavirus. The ELISA kit has the characteristics of high sensitivity, good specificity, high purity and the like, and is high in sensitivity, strong in specificity, simple to operate, short in detection time and strong in clinical practicability.)

1. A SARS-CoV-2 coronavirus IgA antibody, comprising: IgA antibody protein sequence number MNWVRQAPGEGLEWLSYISTSSNNIFYADSVKGRFTVSRDNAKNSLYLQMNSLRDEDTAVYYCAGHTGSNWFDYWGQGTLVTVSSASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY.

2. The use of SARS-CoV-2 coronavirus IgA antibody in an ELISA test kit according to claim 1, comprising the following technical measures:

1) comparing and analyzing the full-length sequence information of all antibodies generated by SARS-CoV-2 coronavirus after invading human body with the internal antibody sequence of a patient without SARS-CoV-2 coronavirus infection to obtain the anti-SARS-CoV-2 coronavirus specific antibody IgA full-length sequence;

2) prokaryotic codon optimization is carried out, the optimized sequence is shown as SEQ ID N0.2, the optimized sequence is embedded into pET32a plasmid, pET32a-IgA prokaryotic expression plasmid is synthesized, pET32a-IgA plasmid is transformed into E.coli BL21(DE3) competence, high-efficiency expression and purification are carried out in E.coli BL21(DE3) strain, and finally purified IgA protein is obtained;

3) identifying whether the population is infected with SARS-CoV-2 coronavirus or detecting whether the human serum contains SARS-CoV-2 coronavirus antigen, using IgA protein as coating antibody and establishing ELISA antigen detection method.

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a SARS-CoV-2 coronavirus IgA antibody and application thereof in an ELISA detection kit.

Background

Coronaviruses (Coronaviruses, Co Vs) belong to the family Coronaviridae (Coronaviridae), including the following genera 4: alpha Coronavir (a-CoV), Beta Coronavir (f 3-CoV), Gamma Coronavir (y-CoV), Delta Coronavir (o-CoV). To date, there are six known human coronaviruses. The two coronaviruses have high infection rate and high fatality rate, and are pathogen severe acute respiratory syndrome coronavirus (SARS-CoV) and middle east respiratory syndrome coronavirus (MERS-CoV) which cause SARS outbreak in Guangdong province in 2003 and MERS outbreak in 2012, and belong to B coronavirus. MERS symptoms typically include fever, cough and shortness of breath, even with the development of pneumonia, with a mortality rate of about 34.4%. Symptoms of SARS usually include fever, chills and body aches, even progress to pneumonia, with a mortality rate of about 9.6%.

Disclosure of Invention

In order to solve the problems, the invention discloses a SARS-CoV-2 coronavirus IgA antibody and application thereof in an ELISA detection kit.

The technical scheme of the invention is as follows: a SARS-CoV-2 coronavirus IgA antibody, IgA antibody protein and its sequence number are

MNWVRQAPGEGLEWLSYISTSSNNIFYADSVKGRFTVSRDNAKNSLYLQMNSLRDEDTAVYYCAGHTGSNWFDYWGQGTLVTVSSASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY*。

A SARS-CoV-2 coronavirus IgA antibody and its application in ELISA detection kit, comprising the following technical measures:

1) comparing and analyzing the full-length sequence information of all antibodies generated by SARS-CoV-2 coronavirus after invading human body with the internal antibody sequence of a patient without SARS-CoV-2 coronavirus infection to obtain the anti-SARS-CoV-2 coronavirus specific antibody IgA full-length sequence;

2) and prokaryotic codon optimization is carried out, the optimized sequence is shown as SEQ ID N0.2, the prokaryotic expression plasmid pET32a-IgA is embedded into plasmid pET32a, prokaryotic expression plasmid pET32a-IgA is synthesized, the plasmid pET32a-IgA is transformed into E.coli BL21(DE3) competence, the efficient expression and the purification are carried out in E.coli BL21(DE3) strain, and finally, the purified IgA protein is obtained.

3) Identifying whether the human population is infected with SARS-CoV-2 coronavirus or detecting whether the human serum contains SARS-CoV-2 coronavirus.

The invention has the advantages that: 1. the antibody used in the invention is anti-SARS-CoV-2 coronavirus specific antibody IgA, after codon optimization, the antibody is embedded into a recombinant antigen pET32a-IgA of a prokaryotic vector pET32a, and the antibody is a soluble antibody and has the characteristics of high sensitivity, good specificity, high purity and the like after induced expression and purification. Meanwhile, pET32a-IgA is immunogenic, and therefore can be used as a coating antibody of a detection kit.

2. The ELISA antigen detection kit prepared by the invention can accurately detect whether a sample contains SARS-CoV-2 coronavirus antigen, and has the advantages of high sensitivity, strong specificity, simple operation and short detection time. Sample 0D for this kit_630nmPositive control 0D_630nm(S/P) to determine whether the sample is positive, and finally determining: when the S/P of the sample to be detected is more than or equal to 0.25, the sample is judged to be positive by SARS-CoV-2 coronavirus antigen; when S/P<0.25, it was judged as negative for SARS-CoV-2 coronavirus antigen. The ELISA kit has the advantages of high sensitivity, strong specificity, simple operation and short detection time.

3. The anti-SARS-CoV-2 coronavirus specific antibody IgA full-length protein is expressed in the supernatant, and has high expression level and strong clinical practicability.

Detailed Description

For the purpose of enhancing an understanding of the present invention, the following detailed description will describe specific embodiments of the present invention, which are given by way of illustration only and are not intended to limit the scope of the present invention.

Example 1: obtaining SARS-CoV-2 coronavirus IgA antibody protein:

1. obtaining of recombinant plasmid

The PCR target amplification is carried out on the anti-SARS-CoV-2 coronavirus specific antibody IgA full-length sequence, and simultaneously the amplified IgA complete sequence is embedded into a pET32a vector to obtain a recombinant plasmid pET32 a-IgA. The recombinant plasmid pET32a-IgA is transformed into E.coli BL21(DE3) competence to obtain the recombinant bacterium pET32 a-IgA.

2. Expression and purification of recombinant proteins

Selecting a single colony, inoculating the single colony in 5ml LB liquid culture medium containing 50 mug/ml ampicillin, carrying out shake culture at 37 ℃ and 200r/min for 8 hours, inoculating the bacterial liquid in 400ml LB liquid culture medium containing 50 mug/ml ampicillin according to the proportion of 1%, and carrying out shake culture at 37 ℃ and 200r/min until OD of the bacterial liquid is obtained₆₀₀When the value is 0.5, IPTG is added to the final concentration of 0.8mmol/L, induction expression is carried out for 3 hours, thalli are collected by centrifugation, the collected thalli are re-suspended by BindingBuffer with the volume of 1/10 of original culture, the thalli are crushed in a low-temperature high-pressure crusher and are repeatedly crushed for 3 times, a thalli lysate lOOR/min is centrifuged for 15 minutes, and a supernatant is collected. The collected supernatant was filtered through a 0.22 μm filter and stored at 2 ℃ for further use.

The affinity chromatography purification comprises the following specific steps: firstly, using a Binding Buffer balance chromatographic column with 5 column volumes, then loading, using a Binding Buffer balance chromatographic column with 10 column volumes after loading is finished, then eluting by using an Elution Buffer, starting to collect protein peaks appearing on a screen of an observation instrument until the peaks are finished, stopping collecting the protein until the peaks are finished, keeping the liquid flow rate at 1.O ml/min in the whole process, dialyzing the collected protein by using a TE Buffer solution for 36 hours, changing dialyzate for 1 time every 12 hours, collecting antibody protein after dialysis is finished, carrying out SDS-PAGE analysis, and simultaneously calculating and analyzing the protein purity by using Image J software, wherein the obtained protein purity is about 95%, and the amino acid sequence of the protein is shown in SEQ ID NO.l; the protein concentration is measured by a ultramicro nucleic acid protein detector, and the obtained protein concentration is about l.88 mg/mL.

3. Immunogenicity detection of recombinant protein pET32a-IgA

In order to further verify the immunogenicity of the obtained IgA full-length protein, Western-blot detection is carried out to show that the IgA full-length protein has biological activity.

Example 2: ELISA detection kit for SARS-CoV-2 coronavirus IgA antibody protein construction

1. Preparation of Positive control

Collecting blood of SARS-CoV-2 coronavirus infection middle and late stage patients, centrifuging at 4000r/min for 10 min after blood is sufficiently coagulated, separating serum, inactivating at 60 deg.C for 30 min, adding thimerosal with final concentration of 0.01%, and storing at-70 deg.C. Diluting the prepared serum by 10 times with protective agent, and filtering and sterilizing with 0.22 μm filter to obtain positive control.

2. Preparation of negative control

Collecting blood of healthy human not infected with SARS-CoV-2 coronavirus, centrifuging at 4000r/min for 10 min after blood is fully coagulated, separating serum, inactivating at 60 deg.C for 30 min, adding thimerosal with final concentration of 0.01%, and storing at-70 deg.C. The prepared serum is diluted by 10 times by using a protective agent, and is filtered and sterilized by a 0.22 mu m filter, so that the negative control is obtained.

3. Preparation of enzyme-labeled plate

The coating concentration of the antibody was determined to be 1:5000 by a matrix method, and the antibody was coated at 2 ℃ for 14 hours. The coating solution was discarded, and 1% Bovine Serum Albumin (BSA) was added as a blocking solution to block the cells at 37 ℃ for 2 hours. And removing the confining liquid, naturally drying, vacuumizing and packaging to obtain the finished product reagent kit enzyme label plate.

4. Preparation of other Components of the kit

Coating liquid: sodium carbonate (Na)₂C0₃) 1.59 g, 2.93g of sodium bicarbonate (NaHC03), adding water for injection to 1000ml, adjusting the pH value to 9.6, and storing at 2-8 ℃ for later use.

Sealing liquid: bovine Serum Albumin (BSA) 5.Og, sucrose 10.0g, sodium chloride (NaCl) 8.5 g, Tween-200.5 ml, and merthiolate sodium 0.10g, adding water for injection to 1000ml, and storing at 2-8 deg.C for use.

A protective agent: collecting potassium chloride (KCl) 0.2g, sodium chloride (NaCl) 8.Og, and potassium dihydrogen phosphate (KH)₂P0₄) 0.27g disodium hydrogen phosphate dodecahydrate (Na)₂HP0₄·12H₂0) 1.42g, Bovine Serum Albumin (BSA) 5.Og, thimerosal 0.lg, Tween 20(Tween-20) 0.5ml, sucrose 2.0g, adding water for injection to 1000ml, and storing at 2-8 deg.C for use.

Sample diluent: sodium chloride (NaCl) 8.Og, disodium hydrogen phosphate dodecahydrate (Na)₂HP0₄·12H₂0)2.9g of potassium dihydrogen phosphate (KH)₂P0₄)0.2g, 0.2g of potassium chloride (KCl), O.lg of thimerosal sodium, and water for injection to 1000 ml.

20 times of concentrated washing solution: tween 20(Tween-20) 10.0ml, sodium chloride (NaCl) 160.0g, disodium hydrogen phosphate dodecahydrate (Na)₂HP0₄·12H₂0)58.0g of potassium dihydrogen phosphate (KH)₂P0₄)4.Og, potassium chloride (KCl) 4.Og, and water for injection to 1000 ml.

Substrate color developing solution: citrate-phosphate buffer (pH 5.0) to prepare 0.lmol/L citrate (C)₆H₈O₇•H₂O) and 0.2mol/L phosphate (Na)₂HP0₄·12H₂0) Mixing 6.1 ml: 6.4ml, preparing TMB stock solution by dissolving TMB in dimethyl sulfoxide (DMSO) to a final concentration of 32mmol/L, diluting TMB stock solution with citrate-phosphate buffer solution at a ratio of 1:20 times, adding polyethylene glycol (PEG) with a final concentration of 7.5mmol/L, lOOmmol/L glucose and 2.94mmol/L H₂O₂After complete dissolution, the mixture is stored in the dark and stored at the temperature of 2-8 ℃ for later use.

Stopping liquid: adding 2.5ml hydrofluoric acid (HF) into 900ml deionized water, diluting to 1000ml, subpackaging, and storing at 28 deg.C.

Example 3: the usage of ELISA detection kit prepared from SARS-CoV-2 coronavirus IgA antibody protein:

sample treatment: taking whole blood, centrifuging at 4000 rpm for 10 minutes after blood coagulation, and collecting supernatant. Blood can also be collected, after coagulation, serum is naturally separated out, and the serum is required to be clear without hemolysis.

Preparing a washing solution: before use, the concentrated wash solution is removed from the kit, allowed to equilibrate to room temperature (20 ℃), shaken, and the precipitate dissolved (preferably by heating in a 37 ℃ water bath for 5 minutes), then diluted 20-fold with distilled water, mixed well, and the diluted wash solution can be stored at 2 ℃ for 7 days.

Preliminary sample dilution: the serum sample to be detected is diluted in a serum dilution plate according to the proportion of 1:40 (for example, 195 mu 1 of sample diluent is added in the serum dilution plate, 5 mu 1 of serum to be detected is added in the serum dilution plate), the positive control and the negative control are diluted in the serum dilution plate according to the proportion of 1:4 (for example, 60 mu 1 of positive control or negative control is added in 180 mu 1 of sample diluent), different samples need to be subjected to tip replacement, and the samples need to be fully mixed in the dilution process.

And taking a proper amount of antibody coated plate, adding 200 mu 1 of washing solution into each hole, washing for 1 time, and patting dry. And then adding 100 mul of the diluted serum to be detected, the positive control and the negative control into the antibody coated plate, wherein 1 hole is formed in the serum to be detected, 2 holes are formed in the positive control and the negative control, and the serum to be detected and the positive control and the negative control are incubated at 37 ℃ for 30 minutes.

2. The liquid in the wells was discarded, 200. mu.l of washing solution was added to the Hunter wells, the washing was repeated 5 times, and the wells were patted dry for the last time.

3. Add 100. mu.1 enzyme label per well and incubate at 37 ℃ for 30 minutes.

4. The well was discarded and washed as in step 2.

5. Adding 100 mu 1 of substrate color development liquid into each hole, and placing the mixture at 20-25 ℃ in a dark place for color development for 10 minutes.

6. Adding 50 mu 1 stop solution into each hole, and reading 0D within 10 minutes_630nmThe value is obtained.

The conditions for the test are as follows: positive control 0D_630nmMean and negative control 0D_630nmThe difference between the average values is not less than 0.8. S is sample 0D_630nmValue, P is a positive control 0D_630nmAverage, N is negative control 0D_630nmAverage value. If the S/P value is more than or equal to 0.25, the sample is judged to be positive; if the S/P value is<0.25, the sample was judged negative.