CN114316039A

CN114316039A - Kit for rapidly detecting viruses and preparation method thereof

Info

Publication number: CN114316039A
Application number: CN202210038830.8A
Authority: CN
Inventors: 陈卫国; 林宣伟
Original assignee: Individual
Current assignee: Baiding Bioengineering Beijing Co ltd
Priority date: 2022-01-13
Filing date: 2022-01-13
Publication date: 2022-04-12
Anticipated expiration: 2042-01-13
Also published as: CN114316039B

Abstract

The invention discloses a kit for rapidly detecting viruses and a preparation method thereof. On one hand, the invention screens a monoclonal antibody capable of specifically recognizing hantavirus NP protein, and the sequences of a heavy chain variable region and a light chain variable region of the monoclonal antibody are shown as SEQ ID NO.2 and SEQ ID NO. 3. The NP protein prepared by combining the monoclonal antibodies and the polyclonal antibodies are prepared into two colloidal gold test strips (antigen detection or antibody detection), the test strips have good specificity and sensitivity, and the test strips are combined for use, so that the detection accuracy can be further improved, and the method is suitable for large-scale popularization and use.

Description

Kit for rapidly detecting viruses and preparation method thereof

Technical Field

The invention belongs to the technical field of virus epidemic disease diagnosis, and particularly relates to a kit for rapidly detecting viruses and a preparation method thereof.

Background

Hantavirus, also known as the renal syndrome hemorrhagic fever virus (HRSV), was first successfully isolated in 1978 from hypsizigus nigra mice. Is distributed in China, Korean peninsula, Russia and other eastern hemisphere areas, and can cause hemorrhagic fever with renal syndrome. Hantaviruses worldwide can be divided into 6 serotypes, i.e., the mouse, rattus, rat, palmar, volar, luteolin, and mouse or mus musculus types. Many wild rodents such as hemimystus nigromaculata are natural hosts of viruses, which have no clinical symptoms but can be transmitted to humans through excretions and secretions. The main clinical manifestations are that after the prodromal symptoms of fever, headache and the like of about 4 days, acute respiratory failure which is characterized by non-cardiogenic pulmonary edema and high fatality rate (52.4-78.0%) occurs, the severe patients die after 3-7 days, and survivors recover quickly without sequela. The pathogenesis of the virus is mainly the direct pathogenic action of the virus, the kidney is an early primary damaged organ, and the virus is a direct factor of kidney injury.

Hantaan virus belongs to Bunyaviridae, is an enveloped segmented negative strand RNA virus, the genome of which comprises L, M, S3 segments respectively encoding L polymerase protein, G1 and G2 glycoprotein, and Nucleoprotein (NP). The NP is the main structural protein of the virus, the immunogenicity is strong, the antibody titer produced by the body is high, the maintenance time is long, the NP plays an important role in the anti-virus immunity, the NP amino acid sequences of different strains and types of viruses are conservative, and carry T cell and B cell antigenic determinants, the NP can induce the body to generate specific humoral immune response and cellular immune response, can be directly combined with the RNA of the in vitro transcription virus and the organelle of the infected cell, plays a role in regulating the virus replication, and is commonly used as a diagnostic protein after HV infection.

Jiro Arikawa et al use baculovirus-expressed viral nucleoproteins for ELISA and can be used for serological monitoring of hantavirus infection. The use of recombinant nucleoproteins and N-terminal deleted nucleoproteins for ELISA and IFA provides a rapid, sensitive, safe diagnostic method for hantavirus infection.

Disclosure of Invention

In view of the defects of the prior art, the invention mainly aims at 2 points, and firstly, a monoclonal antibody of hantavirus nucleoprotein and a preparation method thereof are researched; the second is to research a detection method and a kit for preparing hantavirus by using the NP protein and/or the monoclonal antibody prepared by the invention. In order to achieve the purpose of the invention, the invention is realized according to the following technical scheme:

firstly, the invention prepares a monoclonal antibody aiming at hantavirus NP, and the sequences of a heavy chain variable region and a light chain variable region of the monoclonal antibody are shown as SEQ ID NO.2 and SEQ ID NO. 3.

Further, the present invention further analyzes the sequence of the monoclonal antibody, wherein the heavy chain variable region of the monoclonal antibody comprises CDR1, CDR2, CDR 3; the amino acid sequences of the CDR1, the CDR2 and the CDR3 are respectively shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6. The light chain variable region of the monoclonal antibody comprises CDR1, CDR2 and CDR 3; the amino acid sequences of the CDR1, the CDR2 and the CDR3 are respectively shown in SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO. 9.

Secondly, the present invention develops a kit for detecting hantavirus based on the monoclonal antibody prepared, said kit comprising an effective amount of the monoclonal antibody of claim 1. The kit is a colloidal gold detection test strip, and the test strip comprises a latex pad prepared from a polyclonal antibody, a nitrocellulose membrane, absorbent paper and a PVC (polyvinyl chloride) back plate; the nitrocellulose membrane comprises a detection line prepared by monoclonal antibody and a quality control line prepared by goat anti-rabbit IgG.

Further, the present invention also develops a hantavirus antibody detection kit comprising an effective amount of the monoclonal antibody of claim 1 and hantavirus NP protein. The kit is a colloidal gold detection test strip, and the test strip comprises a latex pad prepared from hantavirus NP protein, a nitrocellulose membrane, absorbent paper and a PVC (polyvinyl chloride) back plate; the nitrocellulose membrane comprises a detection line prepared from hantavirus NP protein and a quality control line prepared from monoclonal antibody.

And thirdly, the two developed kits are combined to obtain the kit for detecting the hantavirus by the antigen-antibody dual method. The combined detection kit has higher accuracy than that of single detection.

Finally, the invention also provides application of the monoclonal antibody in preparing a hantavirus detection reagent. The applications include but are not limited to IFA detection of hantavirus, colloidal gold detection test paper of hantavirus, and hantavirus antibody detection test paper.

Although the NP protein prepared by the invention is the most basic prokaryotic expression, the protein is also suitable for preparing different hantavirus diagnostic reagents, such as colloidal gold, chemiluminescence and other detection kits.

The monoclonal antibody prepared by the invention has good specificity and sensitivity, is suitable for preparing different hantavirus detection reagents, and the detection reagents not only comprise the colloidal gold detection test strip, but also can be an ELISA detection kit and a chemiluminescence detection kit, and can also be applied to detection of IFA, wertern blot and the like in scientific research.

The two colloidal gold test strips prepared by the invention can be simultaneously applied to the antigen and antibody detection of the hantavirus, have strong specificity, high sensitivity, good stability, high detection speed and higher combined detection accuracy, and can be used for early screening and field screening of the hantavirus.

Drawings

FIG. 1 is an SDS-PAGE pattern of purified hantavirus Nucleoprotein (NP) wherein 1 is Marker and 2 is purified NP protein.

FIG. 2 is a SDS-PAGE pattern of two purified monoclonal antibodies, wherein 1 is Marker and 2 is the purified monoclonal antibody.

FIG. 3IFA assay results.

FIG. 4 shows the test results of the test paper, wherein 1, 2 and 3 are respectively adenovirus inactivated virus solution, influenza A inactivated virus solution and influenza B inactivated virus solution.

FIG. 5 shows the test results of the test strip, wherein 1, 2 and 3 are adenovirus positive serum (after inactivation), influenza A positive serum (after inactivation) and influenza B positive serum (after inactivation), respectively.

Detailed Description

The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Example 1: construction of prokaryotic expression vector for expressing hantavirus Nucleoprotein (NP)

The nucleotide sequence of the CDS sequence of NP protein of GenBank: AF159370.1 is subjected to codon optimization, the nucleotide sequence of NP protein after codon optimization (shown as SEQ ID NO. 1) is cloned into a prokaryotic expression vector (PET28a), and 6 His tags are added at the C end. This work was entrusted with the Huada gene.

Example 2: expression and purification of hantavirus Nucleoprotein (NP)

Carrying out shake culture on the induced genetically engineered bacteria (PET28a-NP) expressing NP protein at 37 ℃ and 220rpm for 6h, then harvesting the thallus, collecting the thallus by using a 50mL centrifugal tube, centrifuging for 10min at 4 ℃ and 5000rpm, and discarding the supernatant to retain the thallus; the thalli is resuspended and washed in 20mL PBS solution, centrifuged at 12000rpm at 4 ℃ for 10min, and the thalli is kept after the supernatant is discarded; resuspending the thallus in 20mL of washing solution, carrying out ultrasonic crushing in an ice bath, wherein the ultrasonic frequency is 40%, the ultrasonic frequency is 3s, the pause is 5s, and the ultrasonic time is 8 min; centrifuging at 12000rpm for 10min at 4 deg.C, suspending the thallus in 20ml of washing solution containing a small amount of urea, centrifuging at 12000rpm for 10min at 4 deg.C, and discarding the supernatant to retain the thallus. The recombinant protein was purified using a nickel column HIS purification system from GE, the purification operation is briefly as follows: resuspending the precipitate with 20ml of a lysis solution, centrifuging at 4 ℃ and 12000rpm for 10min, and transferring the supernatant into another centrifuge tube; suspending 1ml of 50% NI-NTA resin into 4ml of lysate, mixing uniformly, and incubating overnight in a shaking table at 4 ℃; adding the mixture of the lysate and the NI-NTA resin suspension into a hollow column with a closed lower end, and collecting effluent liquid; adding 12ml of renaturation solution containing urea, placing the renaturation solution on a shaking table, carrying out ice bath incubation for 6h to renaturate the target protein, and finally flowing out the renaturation solution; eluting with urea-containing eluent for 3 times (12ml, 6ml), placing into shaker at 4 deg.C for 30min for the first time, placing into shaker at 4 deg.C for 15min for the other two times, eluting the target protein, and collecting eluent; concentrating the collected eluent to 3-5 ml by using an ultrafiltration tube, dialyzing in a PBS buffer solution for changing the liquid, subpackaging and storing at-20 ℃ for later use.

10 μ l of the purified NP protein was electrophoretically examined on SDS-PAGE protein gel, showing a clear band at about 49kDa with a purity of 90% or higher (FIG. 1). Protein content was measured using the BCA kit, protein concentration was 1.02 mg/ml.

Example 3: preparation and purification of hybridoma cell expressing NP protein monoclonal antibody

Mouse immunization: the purified NP protein is used as immunogen to immunize female BALB/c mice with age of 8-10 weeks. The immunization method comprises the following steps: first-stage immunization, mixing 100 μ g NP protein and equivalent FCA for each mouse to prepare an emulsifier, and performing subcutaneous multipoint injection and intraperitoneal injection on neck and back; second immunization, 2 weeks after first immunization, FCA is changed into FICA, and the dosage and method are the same as above; the third immunization was performed 2 weeks after the second immunization, and the dose and method were the same as the second immunization. The cells were boosted 1 week prior to fusion by intraperitoneal injection of 100. mu.g of purified NP protein.

Cell fusion: cell fusion was performed 3-5 days after the mice were boosted. The cell fusion procedure is briefly described as follows: killing the positive mouse by breaking the neck, taking splenocytes of the mouse aseptically, fusing the splenocytes with PEG4000 according to the proportion of 5:1 of SP2/0 myeloma cells, stopping fusing by adding RPMI1640 after standing for 1min, centrifuging at room temperature of 1000rpm for 8min, discarding supernatant, adding 50ml of complete RPMI1640 containing 20% HAT newborn bovine serum, and adding the fused cells into a 96-well plate with 100 mul per well. The plates were incubated at 37 ℃ with 5% CO₂Culturing in incubator, and taking complete RPMI1640 containing HT 20% calf serum after 15 days.

Screening cell strains: and carrying out subcloning on the screened positive hybridoma cells, wherein the subcloning adopts a limiting dilution method, original well cells are diluted by an HT (high temperature medium) medium and then are divided into 96-well cell plates, and one original well cell is divided into one plate. After subcloning, the number and status of cells in each well were observed. Wells in which the secreted antibody was stable and as single clone as possible were subcloned were taken for a second subcloning. After three times of subcloning, the positive rate of the detection result of the subcloned plate which is originally monoclonal reaches 100%. 1 hybridoma cell strain capable of stably secreting monoclonal antibody specific to NP protein is obtained.

Example 4: preparation and detection of NP protein monoclonal antibody

Preparing ascites: injecting liquid paraffin into the abdominal cavity of 8-10 week-old healthy BALB/c mice, each 0.5ml, injecting the liquid paraffin into the abdominal cavity 10 days later⁶Hybridoma cell, which is used as mouse 7-10 days laterAnd (3) extracting ascites once every 2 days when the abdomen is extremely swollen, centrifuging the extracted ascites at the temperature of 2-8 ℃ for 10 minutes at 1000rpm, removing upper-layer grease and precipitates, subpackaging the supernatant (5 ml/tube), and storing at-20 ℃ or-70 ℃ for later use.

Antibody purification: the method is characterized in that the method is an octanoic acid-ammonium sulfate precipitation method (particularly, refer to perijade and the like, and the research on a method for purifying mouse ascites IgG monoclonal antibodies, as shown in No. 10 in 2006), after purification, the purity of the purified antibodies is tested by SDS-PAGE, and the result shows that the SDS-PAGE purity of the monoclonal antibodies is more than 90 percent (shown in figure 2); the purified antibodies were subjected to concentration detection using the BCA kit, and the detection results were 5.82mg/ml, respectively.

And (3) measuring the antibody titer: the purified NP protein was added at 0.1. mu.g/well to an ELISA plate and left overnight at 4 ℃. After washing for 3 times, 200. mu.L of blocking solution was added to each well, and the mixture was left at 37 ℃ for 1 hour and washed for 3 times. The prepared antibody was serially diluted 10-fold with PBS, 100. mu.L/well was added to the blocked ELISA plate, and PBS was used as a blank control; incubating for 1h in a 37 ℃ incubator, washing for 3 times, adding an antibody of an HRP-labeled goat anti-mouse (1:5000), 100 mu L/well, incubating for 0.5h in the 37 ℃ incubator, washing for 3 times, adding a freshly prepared substrate solution for 100 mu L/well, incubating for 10min in the 37 ℃ incubator in a dark place, adding 50 mu L/well stop solution, determining OD450, and taking P/N > 2.1 as a positive determination standard (P is the OD450 value of a sample, and N is the OD450 value of a blank control). The results of the assay (Table 1) show that the titer of the monoclonal antibody was 10⁷。

TABLE 1 results of antibody titer measurement

Dilution factor	10³	10⁴	10⁵	10⁶	10⁷	10⁸	10⁹
								OD450 value	1.443	1.354	0.765	0.454	0.201	0.054	0.061
P/N value	21.54	20.21	11.42	6.78	3.00	0.81	0.91

Note: blank OD450 was 0.067.

Monoclonal antibody subclass identification: the mouse monoclonal antibody typing gold-labeled test paper kit of the IsoTrip company is adopted for identification, and the specific steps are operated according to the instruction of the kit. The monoclonal antibody subclass is IgG through detection₁。

Determination of monoclonal antibody sequences: the monoclonal antibody prepared was subjected to the determination of the heavy chain variable region and the light chain variable region by the method of example 7 of the chinese patent (202110793081.5). Through detection and sequencing, the sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown as SEQ ID NO.2 and SEQ ID NO. 3; the determined sequences are analyzed, and the result shows that the amino acid sequences of 3 CDRs in the heavy chain variable region of the monoclonal antibody are respectively shown as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6; the amino acid sequences of 3 CDRs in the light chain variable region are respectively shown as SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO. 9.

Example 5: FITC-labeled monoclonal antibody and detection thereof

The specific test method is disclosed in patent 201710054599.0, which is briefly described as follows: concentrating the monoclonal antibody to a concentration of 10mg/ml, adjusting the pH to 9.0, and storing at 4 ℃ for later use; dissolving 1mg FITC in 0.2ml DMSO, slowly dripping into antibody, reacting at room temperature in dark for 2h, and purifying with Sephadex G25 column after reaction. The purified monoclonal antibody was diluted 1:100 with PBS containing 1% FBS, and IFA detection was performed on Hantaan virus infected mouse lung tissue (identified by IFA, PCR and sequencing). The results show (FIG. 3) that dense green fluorescent particles were seen in the lung tissue of infected mice under fluorescence microscopy, indicating that the monoclonal antibodies prepared according to the invention have good specificity and sensitivity.

Example 6: preparation and detection of colloidal gold detection test strip (antigen)

Preparation of polyclonal antibody: the NP protein prepared by the invention is used for preparing a vaccine (100 mu g/ml of emulsified antigen and adjuvant in equal mass ratio) by using an ISA 201 adjuvant, and 1 ml/rabbit is injected into a new Zealand rabbit at multiple points on the back; boosting once with the same dose and route after 2 weeks interval; boosting once more with the same dose after 2 weeks interval; 7 days after the three-immunization, rabbit blood is collected, centrifuged at 3000rpm for 10min, serum is separated, and the rabbit blood is stored at-20 ℃ for later use. The separated serum was purified with ProteinG pre-packed column for antibody concentration, and after concentration, the antibody concentration was measured with BCA at 2.12mg/ml, and the antibody titer was measured by the antibody titer measuring method of example 4 at a titer of 10⁵Subpackaging and storing at-20 ℃ for later use.

Preparing a test strip: centrifuging 1ml of carboxyl modified latex microspheres (Shanghai brightness, 400nm) at 12000rpm at room temperature for 30min, cleaning with 0.1mol/L MES solution for 3 times, adding 5mmol/L EDC and 2mmol/L NHS, activating at room temperature for 30min, adding 1mg of prepared polyclonal antibody after activation, reacting at 37 ℃ in an incubator for 3h, adding 2% BSA, sealing, and reacting at room temperature for 30 min; the labeled polyclonal antibody is centrifuged at 12000rpm for 30min, resuspended in PBS containing 1% BSA, spread evenly on the treated latex microsphere pad, placed in an oven at 37 ℃ overnight, and stored at room temperature for drying and preservation. Diluting goat anti-rabbit IgG to 0.5mg/ml with PBS buffer solution, diluting monoclonal antibody to 1mg/ml with PBS buffer solution, spraying goat anti-rabbit IgG on the position of a quality control line (C line), spraying monoclonal antibody on the position of a detection line (T line) to prepare a nitrocellulose membrane, placing the nitrocellulose membrane in an oven at 37 ℃ overnight, and drying and storing at room temperature for later use. And (3) assembling the dried nitrocellulose membrane with a latex pad, absorbent paper and a PVC (polyvinyl chloride) back plate, and cutting to obtain the hantavirus rapid detection test strip for detection. In the detection, a sample to be detected is dripped into a detection hole (about 40 mu l), and an equal amount of sample diluent (PBS buffer solution) is immediately added; after sample adding, the test strip is horizontally placed on a desktop, the result is observed for about 8-10 minutes, and if strips appear on both the T line and the C line, the test strip is judged to be positive; if the C line has a strip, the T line has no strip, and the judgment is negative; if the C line has no stripe, the T line is judged to be invalid whether the T line has a stripe or not.

And (3) test strip detection: the prepared test paper is used for detecting negative reference products of adenovirus inactivated virus liquid, influenza A inactivated virus liquid and influenza B inactivated virus liquid respectively, and the detection results (figure 4) show that the detection results of the negative reference products are negative. The prepared test strip is used for detecting NP protein with different concentrations, and the result shows that (table 2) the minimum detection limit of the test strip can reach 2 ng/ml.

TABLE 2 detection results of NP proteins at different concentrations

Concentration of NP protein	8ng/ml	4ng/ml	2ng/ml	1ng/ml
					The result of the detection	+	+	+	-

Note: "+" indicates a positive test result, and "-" indicates a negative test result.

Example 7: preparation of colloidal gold detection test paper strip (antibody) and detection thereof

On the basis of example 5, the latex pad-labeled polyclonal antibody was replaced with the NP protein prepared in the present invention; the T line (detection line) is also replaced by the NP protein prepared by the invention, and the C line (quality control line) is replaced by the monoclonal antibody prepared by the invention; the test paper strip for preparing the double-antigen detection antibody has the same preparation process, detection steps and detection result judgment as those of the test paper strip in the embodiment 5.

And (3) test strip detection: the prepared test strip is used for detecting negative reference adenovirus positive serum (after inactivation), influenza A virus positive serum (after inactivation) and influenza B virus positive serum (after inactivation), and the detection results (shown in figure 5) show that the detection results of the negative reference are negative. The prepared test strip is used for detecting hantavirus positive serum (after inactivation) with different dilutions, and the result shows that the minimum detection limit of the test strip can reach 1:3200 (Table 3).

TABLE 3 detection results of different dilutions of hantavirus positive serum (after inactivation)

Degree of dilution

1:100

1:200

1:400

1:800

1:1600

1:3200

1:6400

The result of the detection

+

-

Example 8: joint detection of two colloidal gold detection test strips

232 collected clinical samples (189 negative samples and 43 positive samples which are all related vaccines inoculated and derived from a certain Hospital clinical laboratory in Guangdong) are detected, and single detection and combined detection (at least one of the samples is positive and both of the samples are negative) are carried out, so that the result shows (shown in table 4), the positive rate of the single detection of the two test strips is over 90%, but the positive rate of the combined detection is 97.7%, and the detection is higher than that of the single detection, and therefore, the combined application of the two test strips can further improve the diagnosis accuracy.

TABLE 4 clinical specimen test results

Test paper strip	Positive rate	Negative rate
			Test paper strip (antigen detection)	39/43＝90.1％	189/189＝100％
Test paper strip (antibody detection)	40/43＝93％	189/189＝100％
			Test paper strip (simultaneous detection)	42/43＝97.7％	189/189＝100％

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

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Claims

1. The monoclonal antibody for detecting the hantavirus is characterized in that sequences of a heavy chain variable region and a light chain variable region of the monoclonal antibody are shown as SEQ ID NO.2 and SEQ ID NO. 3.

2. The monoclonal antibody of claim 1, wherein the heavy chain variable region of said monoclonal antibody comprises CDRs 1, CDR2, CDR 3; the amino acid sequences of the CDR1, the CDR2 and the CDR3 are respectively shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6.

3. The monoclonal antibody of claim 1, wherein the light chain variable region of said monoclonal antibody comprises CDRs 1, CDR2, CDR 3; the amino acid sequences of the CDR1, the CDR2 and the CDR3 are respectively shown in SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO. 9.

4. A kit for detecting hantavirus, said kit comprising an effective amount of the monoclonal antibody of claim 1.

5. The kit according to claim 4, wherein the kit is a colloidal gold test strip, and the test strip comprises a latex pad, a nitrocellulose membrane, absorbent paper and a PVC back plate, which are prepared from a polyclonal antibody; the nitrocellulose membrane comprises a detection line prepared by monoclonal antibody and a quality control line prepared by goat anti-rabbit IgG.

6. A hantavirus antibody detection kit, comprising effective amounts of the monoclonal antibody of claim 1 and hantavirus NP protein.

7. The kit of claim 6, wherein the kit is a colloidal gold test strip, and the test strip comprises a latex pad, a nitrocellulose membrane, absorbent paper and a PVC (polyvinyl chloride) back plate, which are prepared from Hantaan virus NP protein; the nitrocellulose membrane comprises a detection line prepared from hantavirus NP protein and a quality control line prepared from monoclonal antibody.

8. A kit for antigen-antibody dual detection of Hantaan virus, comprising the kit of claim 5 and the kit of claim 7.

9. Use of the monoclonal antibody of claim 1 in the preparation of a hantavirus detection reagent.