CN110343707B

CN110343707B - Hepatitis B core antigen-based HSV (herpes Simplex Virus) typing protein, kit, method and application

Info

Publication number: CN110343707B
Application number: CN201910460579.2A
Authority: CN
Inventors: 王祥喜; 饶子和; 朱凤才; 崔仑标; 郑滨洋; 胡月梅; 张黎; 王康
Original assignee: Institute of Biophysics of CAS
Current assignee: Institute of Biophysics of CAS
Priority date: 2019-05-30
Filing date: 2019-05-30
Publication date: 2021-06-15
Anticipated expiration: 2039-05-30
Also published as: CN110343707A

Abstract

The invention provides a hepatitis B core antigen-based protein for HSV typing, a kit, a method and application. The method for obtaining the protein for typing HSV based on the hepatitis B core antigen comprises the following steps: respectively embedding a section of epitope of the HSV-1 protein epitope and a section of epitope of the HSV-2 protein epitope into an HBCag skeleton gene sequence to form an HSV-1 recombinant sequence and an HSV-2 recombinant sequence, and respectively inserting the HSV-1 recombinant sequence and the HSV-2 recombinant sequence into a prokaryotic expression vector to obtain an HSV-1 recombinant sequence expression vector and an HSV-2 recombinant sequence expression vector; transferring the recombinant sequence expression vector of HSV-1 and the recombinant sequence expression vector of HSV-2 into cells for expression to obtain protein particles; and purification of protein particles. The protein particles obtained by the invention can be very well applied to HSV typing diagnosis.

Description

Hepatitis B core antigen-based HSV (herpes Simplex Virus) typing protein, kit, method and application

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a hepatitis B core antigen (HBV) typing protein, a hepatitis B core antigen based HSV typing kit, a hepatitis B core antigen based HSV typing method and application.

Background

Herpes Simplex Virus (HSV) belongs to the sub-family of the virus a of the family herpesviridae, and the size of the viral plasmid is about 180 nanometers. Clinically, the virus is classified into type 1 and type 2 according to the difference of antigenicity in venereal disease hospitals. Type 1 is mainly obtained from the orolabial lesions, and type 2 can be isolated from the genital lesions. The infection is due to human-to-human contact. The number of infected people from four months to years after the occurrence can reach 50% -90% of the total population, and the virus is the virus which is most easily invaded by people, but only has a part of the clinical morbidity. The disease can be divided into: herpes labialis, herpetic keratitis, herpetic dermatitis, pudendal herpes, and Kaposi disease, and may also be the cause of meningitis and encephalitis.

HSV-1 is more than 90% positive in adults and remains latent for life in sensory ganglia. Approximately 12% -20% of adults in industrialized countries exhibit HSV-2-positivity, and up to 80% of adults in developing countries exhibit HSV-2-positivity. In addition, studies have shown a strong correlation between HSV-2 infection and HIV infection.

The infection rate of HSV-1 in Chinese people is more than 90%, and the infection rate of HSV-2 in Chinese people is more than 10% -30%. Thus, HSV typing diagnostic reagents have great commercial promise in China, and there are companies selling such reagents, e.g., Trinity Biotech in Ireland and some domestic companies. However, current diagnostic agents for typing HSV are not entirely satisfactory.

Disclosure of Invention

The invention provides a hepatitis B core antigen-based protein for HSV typing, a kit and a preparation method thereof. The protein has good sensitivity, specificity and stability when being used for HSV parting diagnosis, basically has no cross antigenicity between HSV-1 and HSV-2, can not be interfered by the skeleton of hepatitis B core antigen, and is very suitable for HSV parting diagnosis.

The invention provides a method for obtaining a hepatitis B core antigen (HSV) typing protein, which comprises the following steps: respectively embedding a section of epitope of the HSV-1 protein epitope and a section of epitope of the HSV-2 protein epitope into an HBCag skeleton gene sequence to form an HSV-1 recombinant sequence and an HSV-2 recombinant sequence, and respectively inserting the HSV-1 recombinant sequence and the HSV-2 recombinant sequence into a prokaryotic expression vector to obtain an HSV-1 recombinant sequence expression vector and an HSV-2 recombinant sequence expression vector; transferring the recombinant sequence expression vector of HSV-1 and the recombinant sequence expression vector of HSV-2 into cells for expression to obtain protein particles; and purification of the protein particles.

In the above method, wherein the one epitope of the HSV-1 protein epitope is 58-93 aa.

In the above method, wherein the epitope of the HSV-2 protein epitope is 545-574 aa.

In the above method, wherein the embedding of a segment of an epitope of an HSV-1 protein epitope and a segment of an epitope of an HSV-2 protein epitope into an HBCag backbone gene sequence, respectively, comprises: respectively embedding a section of epitope of the HSV-1 protein epitope and a section of epitope of the HSV-2 protein epitope between 79-80aa of the HBCag skeleton gene sequence.

The invention also provides protein particles obtained by the above method.

The invention also provides application of the protein particle in HSV typing diagnosis.

The invention also provides an ELISA kit, which comprises the protein particles, wherein the protein particles are coated in the holes of an ELISA plate.

In the above ELISA kit, the coating amount of the protein particles is 5 ng/well.

The invention also provides application of the ELISA kit in HSV typing diagnosis.

The protein particles obtained by the invention are used for HSV typing diagnosis, can obtain very high sensitivity, good specificity and stability, and have obvious advantages and very good application prospect compared with the current HSV typing diagnosis products or modes.

Drawings

FIG. 1A shows the partial sequence of HSV-1 and HSV-2.

FIG. 1B shows a schematic of the molecular construction.

FIG. 2A shows SDS-PAGE and transmission electron microscopy images of HBCag particles.

FIG. 2B shows SDS-PAGE and TEM images of HBCag particles obtained after insertion of HSV-1 specific epitopes (58-93 aa).

FIG. 2C shows SDS-PAGE and TEM images of HBCag particles obtained after insertion of HSV-2 specific epitopes (545-574 aa).

FIG. 3 and FIG. 4 show ROC curves of the test results of ELISA prepared by the present invention, respectively.

FIGS. 5A and 5B show ROC analysis of HSV-1IgG and HSV-2IgG, respectively, of the ELISA of the present invention with some domestic ELISA kits.

Figure 6 shows the results of the ELISA assay of the invention, the results of the Trinity kit assay and the results of the IFA assay.

FIG. 7 shows the cross-reactivity between HSV-1 and HSV-2.

FIG. 8A shows a graph of OD450 as a function of antigen coating amount; FIG. 8B shows OD450 of HBCag as the coating antigen; FIGS. 8C and 8D show OD450 of different sera against different antigens, where 1-12 are HSV-1 epitope antigen and 2-12 is HSV-2 epitope antigen of the present invention, respectively; 1-H-12 is a particle embedded with HSV-1 epitope antigen, and 2-H-12 is a particle embedded with HSV-2 epitope antigen.

Detailed Description

As shown in FIG. 1A and FIG. 1B, the invention selects the 1, 2 type-related specific antigen epitope of HSV-gG protein, and embeds the epitope into the immunodominant region of hepatitis B core antigen monomer, so that the epitope is self-assembled to form particles (particles).

Specifically, first, molecular construction is performed: respectively embedding a section of epitope (58-93aa) of the HSV-1gG protein epitope and a section of epitope (545-574aa) of the HSV-2gG protein epitope into an HBCag skeleton gene sequence (between 79-80 aa) to form respective recombination sequences of HSV-1& 2. The recombinant sequence was inserted into the prokaryotic expression vector pET-28a at two cleavage sites, Nco1 and Xho1, to complete the molecular construction of type 1 and type 2, respectively. Wherein, the sequences of HBCag, HSV-1 and HSV-2 refer to the part of a sequence table, the sequence 1 is the complete sequence of HBCag, and the sequences 2 and 3 are the relevant epitope information of the embedded HSV-1 and HSV-2 respectively.

Then, plasmid transformation was performed: BL21(DE3) competent cells were immediately placed on ice from a-80 ℃ freezer and allowed to thaw for 5-10 minutes. To the competent cells, 1-2. mu.l of plasmid was added and placed back on ice for 30 min. It was then subjected to a water bath at 42 ℃ for 90 seconds. Again on ice for 2 minutes, 800. mu.l LB medium was added. Then, the cells were cultured at 37 ℃ and 220rpm in a constant temperature shaking incubator for about 1 hour. 100 mul of the broth was aspirated and spread evenly on solid petri dishes containing kanamycin resistance. The solid dish was placed in an incubator at 37 ℃ and incubated overnight.

Then, test expression was performed: a single strain was picked from a solid culture dish, inoculated into 5ml of a liquid medium (containing kanamycin at a final concentration of 50. mu.g/ml) and cultured at 37 ℃ and 220rpm to an OD of about 0.6, Isopropylthiogalactoside (IPTG) was added to induce expression of the target protein at a final concentration of 1mM, and the culture was continued at 37 ℃ and 220rpm for 5 hours. Centrifuging the cultured bacterial solution at 4000rpm for 20min, and adding 800 μ l PBS (preparation method: NaCl 8.0g, KCl0.2g, Na)₂HPO₄ 1.44g，KH₂PO₄0.24g, adding distilled water to 1000ml, adjusting pH to 7.4), resuspending the bacteria, and ultrasonically breaking the bacteria (power 200w, ultrasonic 4s, interval 8 s). After crushing, 12000rpm, centrifuging for 5min, taking out the supernatant, re-suspending the precipitate with 800 μ l PBS, and respectively sampling the supernatant and the precipitate re-suspension for SDS-PAGE identification to obtain the target protein which is determined as soluble expression.

Expression of the virus-like particles is then performed: the bacterial liquid 1:100 of the test expression is inoculated in 1L of culture medium (containing kanamycin and the final concentration is 50 mu g/ml), shaking culture is carried out at 37 ℃ until the OD value is about 0.6, IPTG (the final concentration is 1mM) is added for induction, and culture is carried out at 16 ℃ and 220rpm for 16-18 h.

Purification of virus-like particles: the cultured bacterial solution is centrifuged at 3800rpm and 4 ℃ for 40min, the thalli are suspended by using a proper amount of conventional PBS buffer, and then the thalli are crushed by ultrasonic waves (the power is 200W, the ultrasonic waves are carried out for 4s, the interval is 8s, and 200 cycles are carried out). After the crushing is finished, centrifuging at 12000rpm and 4 ℃ for 20min, taking the supernatant after centrifugation, carrying out saturated ammonium sulfate precipitation on the supernatant, dropwise adding saturated ammonium sulfate into the supernatant under stirring in an environment at 4 ℃ until the supernatant becomes turbid, and continuously stirring for 0.5 h. After stirring, the mixture was centrifuged at 12000rpm at 4 ℃ for 10min, and after centrifugation, the pellet was suspended in 1-2ml of PBS. And then carrying out sucrose density gradient centrifugation, wherein the sucrose gradient is 15-45%, centrifuging at 30000rpm and 4 ℃ for 5h after sample loading, carrying out layered sampling after centrifugation, taking 600 ul/tube from top to bottom in an EP tube, and storing in a refrigerator at 4 ℃.

Identification of virus-like particles: the concentration of the obtained virus-like particles is determined, and the virus-like particles are preliminarily identified through SDS-PAGE electrophoresis, and then the morphology of the virus-like particles is observed through an electron microscope, so that the expected virus-like particles are formed. Wherein, FIG. 2A shows SDS-PAGE and TEM image of HBCag particles, FIG. 2B shows SDS-PAGE and TEM image of HBCag particles obtained after embedding HSV-1 specific epitope (58-93aa), and FIG. 2C shows SDS-PAGE and TEM image of HBCag particles obtained after embedding HSV-2 specific epitope (545-574 aa). Wherein, in the SDS-PAGE picture, the left lane is marker, and the right lane is the target protein lane.

Then, the polypeptide obtained by the selected HSV-1 and HSV-2 specific epitope and the particle obtained by embedding the specific epitope into HBCag can be fixed in a substrate, and an ELISA plate is prepared for detection. It should be understood that the following indirect ELISA detection method is merely an illustrative example, and any suitable structure of ELISA plate may be prepared by any suitable method. The establishment of the indirect ELISA detection method (IgG) included: coating: diluting the antigen with PBS, coating a 96-well enzyme label plate according to 0.05 mu g/ml, coating 100 mu l/well and coating overnight; and (3) sealing: blocking with 1% BSA solution (PBS) 300. mu.l/well at 37 deg.C for 2 h; incubating the primary antibody: the serum to be tested is prepared according to the following steps of 1: 21, the diluted solution is 1% BSA solution, and a blank 1-well, negative and positive control (TRINIYT detection kit) is setThe negative and positive control provided in (1), namely HSV-1 or HSV-2 negative and positive serum, the same applies below) each 2 holes, 100 ul/hole of the diluted serum sample to be detected is added into the corresponding reaction hole, and the reaction is carried out for 30min at 37 ℃; washing the plate: washing the plate with a plate washing machine, PBST buffer solution (preparation method: NaCl 8.0g, KCl0.2g, Na)₂HPO₄ 1.44g，KH₂PO₄0.24g, Tween-201 ml, adding distilled water to 1000ml, adjusting pH to 7.4) adding 300 ul/well into each well, shaking the plate for 10s, washing for 5 times, and drying; incubation of secondary antibody: diluting enzyme-labeled secondary antibody at a ratio of 1:3000, adding 100 ul/hole of 1% BSA solution into each hole, and reacting at 37 deg.C for 30 min; washing the plate: washing the plate with a plate washing machine, adding 300ul PBST buffer solution into each hole, shaking the plate for 10s, washing for 5 times, and then drying by beating; color development: adding 100ul of 3,3',5,5' -Tetramethylbenzidine (TMB) color development solution into each well, and reacting at room temperature for 8 min; and (4) terminating: adding 100ul of 2mol/L sulfuric acid solution into each hole, and stopping reaction; reading: and detecting the light absorption value of each hole by using a single wavelength of 450nm of a microplate reader. The reaction conditions shown in Table 1 may be used, but of course, other suitable conditions may be used.

TABLE 1

Thereafter, over 1000 sera were tested using the Trinity HSV typing test kit, known as the industry standard. Distinguishing which are positive and which are negative; a portion of the sera was then selected and manually divided into two groups: one set was used for detection of HSV type 1 and the other set was used for detection of HSV type 2. The procedures were performed according to the instructions described in the kit, and the results are shown in table 2:

TABLE 2

HBCag particles embedded in HSV-1 or HSV-2 epitopes were then coated in ELISA plates using the previously described methods and serum was tested using the procedures described previously. Finally, carrying out ROC curve analysis on the obtained data by using Medcalc software, and calculating Kappa coefficients of the reaction result of the HBCag particles embedded into the HSV-1 or HSV-2 epitope and the Trinity reaction result.

The results of the determination of HSV-1 are shown in Table 3 and FIG. 3, and the results of the determination of HSV-2 are shown in Table 3 and FIG. 4.

TABLE 3

HSV-1	Statistics of	HSV-2	Statistics of
				Cut-off	0.148	Cut-off	0.16
Sensitivity of the probe	93.75％(60/64)	Sensitivity of the probe	98.44％(23/24)
				Specificity of	100.00％(24/24)	Specificity of	100.00％(64/64)
AUC	0.990	AUC	0.999
				95% confidence interval	0.940-1.000	95% confidence interval	0.956-1.000
P	＜0.0001	P	＜0.0001
				Kappa	0.891	Kappa	0.972

As is clear from Table 3, the sensitivity of ELISA prepared by the present invention was more than 93%, the specificity was close to 100%, AUC was 0.99 or more, and Kappa value was 0.891 or more. Generally, a closer AUC and Kappa value to 1 indicates that the results are more nearly identical, and thus, the results are still more stable and desirable.

In addition, the applicant compares the results of the kit of the invention with the results obtained by parallel experiments of some existing kits, on one hand, the results obtained by cut-off values recommended by the kit per se are compared, and on the other hand, the applicant also performs ROC curve analysis on the data of the products and then compares the data with the data of the products of the invention. As shown in FIGS. 5A and 5B, the results both show that the kit prepared by the present invention is significantly superior to some existing kits.

Thereafter, 78 sera identified as 1/2 type double negative by Trinity assay were judged by gold-labeled indirect Immunofluorescence (IFA). The method comprises the following operation steps: 1. spreading the Vero cells in a 96-well plate, respectively using HSV-1 and HSV-2 to infect the cells (the mass ratio of the HSV-1 or HSV-2 to the Vero cells is 1: 1000), and setting a blank control (only inoculating the cells and not inoculating viruses); 2. after cytopathic effect, sucking out supernatant, fixing with 80% ethanol at-20 deg.C for 1-2 hr, pouring off ethanol, and storing at-20 deg.C; 3. washing 96-well plates inoculated with HSV-1 and HSV-2 and blank plates with PBS buffer (containing 0.05% Triton) at 600rpm for 3min and 3 times; 4. 78 parts of serum were mixed according to 1: 21, diluting the mixture according to the proportion of 0.05 percent Triton in PBS, setting blank and positive control holes, diluting the mixture, respectively adding the diluted mixture into three 96-well plates, and reacting the mixture for 2 hours at 37 ℃; 5. removing the liquid from the ELISA plate, washing with PBS buffer (containing 0.05% Triton) at 600rpm for 3min, and washing for 5 times; 6. diluted FITC-labeled anti-human IgG secondary antibody was added to each well at a dilution ratio of 1:100, the diluent is PBS containing 0.05 percent Triton, the reaction is carried out for 1 hour at room temperature, and the whole process is carried out under the condition of keeping out of the sun; 7. removing liquid from the ELISA plate, washing with PBS buffer (containing 0.05% Triton) at 600rpm for 3min, washing for 5 times, and keeping the whole process in dark; 8. and (5) observing by using a fluorescence microscope, and counting the results. In addition, the measurement was also carried out by the ELISA kit of the present invention according to the aforementioned method.

The results are shown in FIG. 6, in which 50% of the sera were positive, i.e., the sensitivity of Trinity was not high enough, and 39 sera that should be positive were judged as negative by mistake. The applicant then tested these sera with ELISA plates prepared by coating them with the proteins of the invention, and showed a match of type 1, type 2 with IFA of 64% and 53%, respectively, and a negative match of 100%. That is, the results of determination of IFA as negative, the present invention and Trinity were negative, but 39 sera determined as positive by IFA were not detected by Trinity, while the methods of

types

1 and 2 of the present invention were able to detect 11 and 3, respectively, which indicates that the ELISA kit of the present invention had higher sensitivity than Trinity.

To demonstrate that the method of the present invention does not cross-react between

types

1 and 2, the applicants selected 56 positive type 1 and negative type 2 sera and performed an indirect ELISA on the type 1 and type 2 particles of the present invention. Selecting 56 parts of HSV1-IgG (+) and HSV2-IgG (-) serum, and respectively reacting the serum with HSV-1 epitope antigen (1-H-12) and HSV-2 epitope antigen (2-H-12) to test whether the serum has cross reactivity.

The specific operation steps are as follows: coating: diluting antigen (1-H-12, 2-H-12) with PBS, coating 96-well enzyme label plate according to 0.05 μ g/ml, 100 μ l/well, coating overnight; and (3) sealing: blocking with 1% BSA solution (PBS) 300. mu.l/well at 37 deg.C for 2 h; incubating the primary antibody: the serum to be tested is prepared according to the following steps of 1: 21, diluting with a 1% BSA solution, setting blank 1 hole and 2 holes of negative and positive controls (negative and positive controls provided in a TRINIYT detection kit, namely HSV-1 or HSV-2 negative and positive serum), adding 100 ul/hole of the diluted sample to be detected into the corresponding hole, and reacting at 37 ℃ for 30 min; washing the plate: washing the plate with a plate washing machine, adding PBST buffer solution into each well at a concentration of 300 ul/well, shaking the plate for 10s, washing for 5 times, and then drying by beating; incubation of secondary antibody: diluting enzyme-labeled secondary antibody at a ratio of 1:3000, adding 100 ul/hole of 1% BSA solution into each hole, and reacting at 37 deg.C for 30 min; washing the plate: washing the plate with a plate washing machine, adding 300ul PBST buffer solution into each hole, shaking the plate for 10s, washing for 5 times, and then drying by beating; color development: adding 100ul of TMB color developing solution into each hole, and reacting at room temperature for 8 min; and (4) terminating: adding 100ul of 2mol/L sulfuric acid solution into each hole, and stopping reaction; reading: and detecting the light absorption value of each hole by using a single wavelength of 450nm of a microplate reader.

As shown in FIG. 7, the crossing rate of the type 1-positive serum with the type 2 antigen was only 3.57%.

Furthermore, it was confirmed that: the framework protein of HBCag has no influence on the actual detection result. Firstly, 4 parts of positive serum and negative serum of HBCag are respectively selected, and indirect ELISA detection is carried out on hepatitis B core antigen HBCag with different coating amounts. 1-H-12/1-12, 2-H-12/2-12 ELISA detection experiment steps: selecting serum HBCag (+)/HSV-1(-), HBCag (+)/HSV-1(+), HBCag (+)/HSV-2(-), and HBCag (+)/HSV-2 (+); coating: diluting antigen (1-H-12/1-12; 2-H-12/2-12) with PBS, coating 96-well enzyme label plate at 0.05 μ g/ml, coating 100 μ l/well, and coating overnight; and (3) sealing: blocking with 1% BSA solution (PBS) 300. mu.l/well at 37 deg.C for 2 h; incubating the primary antibody: the serum to be tested is prepared according to the following steps of 1: 21, diluting with a 1% BSA solution, setting blank 1 hole and 2 holes of negative and positive controls (negative and positive controls provided in a TRINIYT detection kit, namely HSV-1 or HSV-2 negative and positive serum), adding 100 ul/hole of the diluted sample to be detected into the corresponding reaction hole, and reacting at 37 ℃ for 30 min; washing the plate: washing the plate with a plate washing machine, adding PBST buffer solution into each well at a concentration of 300 ul/well, shaking the plate for 10s, washing for 5 times, and then drying by beating; incubation of secondary antibody: diluting enzyme-labeled secondary antibody at a ratio of 1:3000, adding 100 ul/hole of 1% BSA solution into each hole, and reacting at 37 deg.C for 30 min; washing the plate: washing the plate with a plate washing machine, adding 300ul PBST buffer solution into each hole, shaking the plate for 10s, washing for 5 times, and then drying by beating; color development: adding 100ul of TMB color developing solution into each hole, and reacting at room temperature for 8 min; and (4) terminating: adding 100ul of 2mol/L sulfuric acid solution into each hole, and stopping reaction; reading: and detecting the light absorption value of each hole by using a single wavelength of 450nm of a microplate reader.

HBCag coating amount verification experiment steps: selecting 4 parts of HBCag negative serum (23-7.15, 103-7.14, 102-7.16 and 9-7.17) and 4 parts of positive serum (73-7.16, 55-7.16, 161-7.16 and 75-7.16); coating: diluting antigen (HBCag) with PBS, coating 96-well enzyme label plate with concentration of 0.05, 0.25, 0.5, 1, 2, 5 μ g/ml, coating 100 μ l/well, and coating overnight; and (3) sealing: blocking with 1% BSA solution (PBS) 300. mu.l/well at 37 deg.C for 2 h; incubating the primary antibody: the serum to be tested is prepared according to the following steps of 1: 21, diluting the sample at a dilution ratio of 1% BSA solution, setting blank 1 hole and 2 holes for each of negative and positive controls (negative and positive controls provided in the HBCag detection kit, namely HBCag negative and positive serum), adding 100 ul/hole of the diluted sample to be detected into the corresponding reaction hole, and reacting at 37 ℃ for 30 min; washing the plate: washing the plate with a plate washing machine, adding PBST buffer solution into each well at a concentration of 300 ul/well, shaking the plate for 10s, washing for 5 times, and then drying by beating; incubation of secondary antibody: diluting enzyme-labeled secondary antibody at a ratio of 1:3000, adding 100 ul/hole of 1% BSA solution into each hole, and reacting at 37 deg.C for 30 min; washing the plate: washing the plate with a plate washing machine, adding 300ul PBST buffer solution into each hole, shaking the plate for 10s, washing for 5 times, and then drying by beating; color development: adding 100ul of TMB color developing solution into each hole, and reacting at room temperature for 8 min; and (4) terminating: adding 100ul of 2mol/L sulfuric acid solution into each hole, and stopping reaction; reading: and detecting the light absorption value of each hole by using a single wavelength of 450nm of a microplate reader.

Reaction of HBCag-positive serum with HBCag Experimental procedure: selecting 33 parts of HBCag positive serum; coating: diluting antigen (HBCag) with PBS, coating 96-well enzyme label plate according to 0.05 μ g/ml, 100 μ l/well, coating overnight; and (3) sealing: blocking with 1% BSA solution (PBS) 300. mu.l/well at 37 deg.C for 2 h; incubating the primary antibody: the serum to be tested is prepared according to the following steps of 1: 21, diluting the sample at a dilution ratio of 1% BSA solution, setting blank 1 hole and 2 holes for each of negative and positive controls (negative and positive controls provided in the HBCag detection kit, namely HBCag negative and positive serum), adding 100 ul/hole of the diluted sample to be detected into the corresponding reaction hole, and reacting at 37 ℃ for 30 min; washing the plate: washing the plate with a plate washing machine, adding PBST buffer solution into each well at a concentration of 300 ul/well, shaking the plate for 10s, washing for 5 times, and then drying by beating; incubation of secondary antibody: diluting enzyme-labeled secondary antibody at a ratio of 1:3000, adding 100 ul/hole of 1% BSA solution into each hole, and reacting at 37 deg.C for 30 min; washing the plate: washing the plate with a plate washing machine, adding 300ul PBST buffer solution into each hole, shaking the plate for 10s, washing for 5 times, and then drying by beating; color development: adding 100ul of TMB color developing solution into each hole, and reacting at room temperature for 8 min; and (4) terminating: adding 100ul of 2mol/L sulfuric acid solution into each hole, and stopping reaction; reading: and detecting the light absorption value of each hole by using a single wavelength of 450nm of a microplate reader.

The results are shown in fig. 8A to 8D, and show that the negative control and the negative serum are specifically responsive to different coating amounts, while the positive serum OD value shows a two-fold decrease with a two-fold decrease of the coating amount, indicating specific binding between them, while when the coating amount is 25 ng/well or less, the OD value is substantially unchanged and almost the same as that of the negative serum, so that the applicant believes that the antibody specific to the hepatitis b core antigen does not substantially recognize the hepatitis b core antigen in the case of the coating amount of less than 10ng, whereas the coating amount of the present invention is 5 ng/well; for further verification, the applicant carried out ELISA detection on HBCag by using 33 parts of HBCag positive serum, and the results are negative; meanwhile, the applicant further divides HBCag positive serum into HBCag positive HSV1 positive HBCag positive HSV1 negative, HBCag positive HSV2 positive and HBCag positive HSV2 negative, and carries out 1-H-12/1-12 on the two groups respectively; and 2-H-12/2-12, and the results show that the negative and positive results are normal. Therefore, it can be known that the framework protein of HBCag has no influence on the actual detection result.

Subsequently, several hundred sera were tested, and Trinity screened 8 boxes of sera (704) were selected and reacted with 1-H-12 and 2-H-12, respectively, as described previously, and the procedure was not repeated here. The measurement results are shown in Table 4.

TABLE 4

As can be seen from Table 4, these several important parameters were maintained at very high levels, indicating good reproducibility and high stability.

In addition, applicants have selected certain sera for both intra-batch and inter-batch repeat, and have obtained parameters of specific standard deviations and dispersion values. 5 parts of HSV-1&2IgG negative serum and positive serum screened by TRINITY are selected, and each serum is repeatedly determined for 7 times for batch test. Similarly, 5 aliquots of each of the TRINITY-screened HSV-1&2IgG negative and positive sera were selected and assayed 4 times for each serum and tested between batches. The assay experimental procedures were as previously described and are not repeated here. Then, the mean, standard deviation, and coefficient of variation are calculated using the obtained data.

The results for the intra-batch replicates of HSV-1IgG are shown in Table 5 and the results for the intra-batch replicates of HSV-2IgG are shown in Table 6. Results for inter-batch replicates of HSV-1IgG are shown in Table 7 and results for inter-batch replicates of HSV-2IgG are shown in Table 8.

TABLE 5

TABLE 6

TABLE 7

TABLE 8

Generally, a dispersion value of less than 10% is desirable. The results in tables 5 to 8 also show that the established method of the invention has good reproducibility.

Therefore, the results prove that the ELISA provided by the invention has good measurement repeatability, and the potential background interference of the framework protein of HBCag and the potential cross reaction between

types

1 and 2 are eliminated, so that the protein provided by the invention and the kit prepared by the protein can be well used for typing diagnosis of HSV-IgG.

Sequence listing

<110> institute of biophysics of Chinese academy of sciences

<120> hepatitis B core antigen-based protein, kit, method and application for HSV typing

<130> HP181098LZ

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 444

<212> DNA

<213> human

<400> 1

atggacatcg atccgtacaa ggaattcggt gcgaccgtgg agctgctgag ctttctgccg 60

agcgacttct ttccgagcgt tcgtgacctg ctggataccg cgagcgcgct gtatcgtgaa 120

gcgctggaaa gcccggagca ctgcagcccg caccacaccg cgctgcgtca ggcgattctg 180

tgctggggtg aactgatgac cctggcgacc tgggtgggcg ttaacctgga ggacagccgt 240

gatctggtgg ttagctacgt gaacaccaac atgggcctga aattccgtca actgctgtgg 300

tttcacatca gctgcctgac cttcggtcgt gaaaccgtta tcgagtacct ggttagcttt 360

ggcgtttgga ttcgtacccc gccggcgtat cgtccgccga acgcgccgat tctgagcacc 420

ctgccggaaa ccaccgtggt ttaa 444

<210> 2

<211> 108

<212> DNA

<213> human

<400> 2

agcccgaccg cgattagcct gaccaccccg gatcacaccc cgccgatgcc gagcattggc 60

ctggaggaag aggaagagga agagggtgcg ggcgacggtg aacatctg 108

<210> 3

<211> 90

<212> DNA

<213> human

<400> 3

gcgccgccgg gcagcccggc tccgccgccg ccggaacacc gtggtggccc ggaggaattt 60

gagggtgcgg gtgacggtga accgccggag 90

Claims

1. A method for obtaining a hepatitis b core antigen based protein for HSV typing comprising:

respectively embedding a section of epitope of the HSV-1 protein epitope and a section of epitope of the HSV-2 protein epitope into an HBCag skeleton gene sequence to form an HSV-1 recombinant sequence and an HSV-2 recombinant sequence, and respectively inserting the HSV-1 recombinant sequence and the HSV-2 recombinant sequence into a prokaryotic expression vector to obtain an HSV-1 recombinant sequence expression vector and an HSV-2 recombinant sequence expression vector;

transferring the recombinant sequence expression vector of HSV-1 and the recombinant sequence expression vector of HSV-2 into cells for expression to obtain protein particles; and

purifying the protein particles;

wherein the section of the HSV-1 protein epitope is 58-93aa, and the section of the HSV-1 protein epitope is encoded by a sequence 2;

wherein the section of the HSV-2 protein epitope is 545-574aa, and the section of the HSV-2 protein epitope is coded by a sequence 3;

wherein, respectively embedding a section of epitope of HSV-1 protein epitope and a section of epitope of HSV-2 protein epitope into HBCag skeleton gene sequence comprises: respectively embedding a section of epitope of the HSV-1 protein epitope and a section of epitope of the HSV-2 protein epitope between 79-80aa of an HBCag skeleton gene sequence, wherein the HBCag skeleton gene sequence is shown as a sequence 1.

2. Protein particles obtained by the method of claim 1.

3. Use of the protein particle of claim 2 in the preparation of a kit for HSV typing diagnosis.

4. An ELISA kit comprising:

the protein particle of claim 2, coated in a well of an ELISA plate;

wherein the coating amount of the protein particles is 5 ng/hole.