KR20180116568A - Diagnosis kit for hepatitis a comprising hav vp1 antigen - Google Patents

Abstract

The present invention relates to a composition for diagnosing hepatitis A which comprises VP1 antigens of HAV; and a diagnosis kit for hepatitis A using the same. By using the diagnosis kit of the present invention, the diagnosis on the infection by HAV can be quickly and easily performed using only a small amount of specimens without the need for additional equipment or skilled technicians, thereby advancing the treatment of hepatitis A.

Description

[0001] DESCRIPTION [0002] DIAGNOSIS KIT FOR HEPATITIS A COMPRISING HAV VP1 ANTIGEN [0003]

The present invention relates to a hepatitis A diagnostic kit capable of detecting an HAV VP1 antigen, and more particularly to a hepatitis A diagnostic kit comprising a VP1 antigen of HAV.

There are five types of hepatitis viruses, A, B, C, D, and E, and only hepatitis A and E viruses are known to cause acute hepatitis without progressing to chronic hepatitis.

Hepatitis A virus (HAV) is a non-enveloped virus with a diameter of 27 to 32 nm belonging to the Enterovirus family with picornaviridae . The genome consists of about 7.5 kb of single stranded RNA. Although HAV is known to cause human disease only, it is detected that it is detected in animal feces through environmental pollution.

Hepatitis A progresses to acute hepatitis at the onset of the disease, but there is a need for rapid diagnosis due to the fact that no preventive vaccine has been developed yet. Diagnostic kits have already been developed using genes encoding structural proteins of HAV. However, the conventional hepatitis A diagnostic tests were difficult to test with a small amount of specimens, and the results of the tests were unsatisfactory. Therefore, there is a need for a diagnostic kit capable of early diagnosis of HAV infection even with a small amount of specimens.

Korean Patent No. 10-1648106

Thus, the present inventors have provided a diagnostic kit capable of quickly and easily diagnosing hepatitis A virus infection with only a small amount of a sample. Accordingly, the present invention relates to a diagnostic kit capable of detecting the VP1 antigen of HAV present in a specimen and diagnosing whether hepatitis A occurs.

It is an object of the present invention to provide a composition for the diagnosis of hepatitis A comprising the VP1 antigen of HAV (Hepatitis A Virus).

In one embodiment, the VP1 antigen may be a protein of SEQ ID NO: 2 or an immunologically equivalent protein thereof.

Another object of the present invention is to provide a hepatitis A diagnostic kit comprising the diagnostic composition.

In one embodiment, it may be one selected from the group consisting of an RT-PCR kit, an immunostrip, a biochip, and an ELISA diagnostic kit.

In one embodiment, it may be an immunological strip.

As described above, by using the hepatitis A diagnostic kit according to the present invention, infection of the hepatitis A virus can be diagnosed within a short time of about 10 minutes. Further, the diagnostic kit of the present invention can easily and economically diagnose infection of HAV virus by using the blood, feces and other body fluids of the subject, without the expensive sample, the equipment or the skilled skill.

FIG. 1 shows an overall strategy for expressing the VP1 antigen protein by cloning a gene encoding the VP1 antigen of HAV.
2 shows the sequences of the DNA sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2) for cloning the VP1 gene of HAV and the PCR primer set (SEQ ID NOs: 3 and 4) for the cloning.
FIG. 3 shows the result of agarose electrophoresis of the VP1 gene of the HAV PCR amplified from a clone purchased from Origene (left photograph), and the PCR amplified VP1 gene was cloned into the pET28a vector and then treated with restriction enzyme The result (right picture) is shown. It was confirmed that the VP1 gene was contained in the cloning vector by a band of about 900 bp.
Figure 4 shows the result of colony PCR of Escherichia coli transformed with the vector cloned with the VP1 gene. As a result of the above PCR amplification, it was found that VP1 gene was contained in Escherichia coli of transformed colonies.
5 shows the result of SDS gel electrophoresis of the VP1 protein of HAV expressed from the transformed E. coli. As shown in FIG. 5, the presence of VP1 protein induced by IPTG was confirmed.
FIG. 6A shows the composition of the immunodiagnostic strip and the predicted diagnosis result, and FIG. 6B shows the composition of the test antibody used in the immunostrip of the present invention, the test antigen, and the actual diagnosis result. As shown in FIG. 6B, using the immunostimulator of the present invention, the HAV infection in the test sample could be successfully diagnosed.

It is an object of the present invention to provide a composition for the diagnosis of hepatitis A comprising the VP1 antigen of HAV.

In one embodiment, the VP1 antigen may be a protein of SEQ ID NO: 2 or an immunologically equivalent protein thereof.

The term " immunologically equivalent protein " as used herein refers to a protein of the polypeptide, such as, for example, replacing an amino acid with an amino acid having similar properties (e.g., polarity, hydrogen bonding potential, acidity, basicity, hydrophobicity, The corresponding amino acid residues of the protein or enzyme have been changed without altering the overall structure and function.

The " immunologically equivalent protein " is at least 60%, preferably at least 75%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95% Has a 95% or greater amino acid identity and includes polypeptides that have the same or substantially similar properties or functions as the compared native protein or parent protein.

In one embodiment, the VP1 antigen protein of the HAV may be prepared by PCR amplification of the DNA sequence encoding the VP1 antigen, cloning into an expression vector, protein expression, isolation and purification.

Another object of the present invention is to provide a hepatitis A diagnostic kit comprising the diagnostic composition.

In one embodiment, one can be selected from the group consisting of RT-PCR kits, immunostimulatory strips, biochips, and ELISA diagnostic kits, and can be preferably immunostatic strips.

In one embodiment, the diagnosis of hepatitis A by the immunostrip may take 10 minutes.

Embodiments of the present invention will now be described, which are merely examples of the invention and have not been described for the purpose of limiting the claims of the present invention.

Example

≪ Materials and methods >

1. Preparation of VP1 antigen of HAV

(1) securing HAV gene source and preparing primer set

The genotype of the hepatitis A virus (HAV) was obtained by purchasing the HAV-VP1 gene cloned in pCMV-myc-DDK-tagged plasmid from Origene (see FIG. HAV-VP1 DNA sequence information (SEQ ID NO: 1) of (NP_740552) are NCBI site: were confirmed in (http... Www ncbi nlm .nih gov), for PCR amplification of the region to be subcloned from the VP1 gene sequences A set of primers (SEQ ID NOS: 3 and 4) containing appropriate restriction enzyme recognition sites were constructed. The DNA sequence of the VP1 gene, its amino acid sequence and PCR primer set sequence are as shown in FIG. 2 and the sequence listing. Specifically, the 19th to 39th and 873th to 896th sequences in the DNA sequence of SEQ ID NO: 1 are the HAV-NS3-EcoR-S1 and HAV-NS3-Hind-AS1 primer binding sequences, Lt; / RTI > In addition, the amino acid sequence of SEQ ID NO: 2 is the VP1 antigen sequence expressed by the above cloning sequence and used in the diagnostic kit of the present invention.

(2) PCR reaction

Using the primer set of SEQ ID NO: 3, a PCR reaction was performed on the plasmid into which the VP1 gene was inserted. The PCR reaction was performed by mixing 1 μl each of 10 ng DNA substrate, 10 pmol of primer, 1 μl of 2 mM dNTP, 1 μl of 1 ml / mg BSA, 2.5 μl of Mg _² 10 × buffer and 1 μl of 1 U Taq polymerase, , 52 ° C to 58 ° C for 45 seconds, and 72 ° C for 45 seconds for 30 cycles. Then, PCR products were confirmed on 1.2% agarose gel.

(3) Subcloning using pET-28a vector

Each PCR product and pET-28a vector were digested with appropriate restriction enzymes and ligated together using a ligation kit (Enzynomics). The ligation reaction was carried out by reacting at 16 DEG C for 16 hours. XL-1 Blue Escherichia coli was transformed with the above cloned vector, and the transformed Escherichia coli was plated on LB solid medium containing 100 μg / ml of kanamycin antibiotic and cultured, and white transformant colonies were selected .

(4) Colony PCR

The transformant colonies were taken, and the introduction of the VP1 gene was confirmed by plasmid extraction or colony PCR. For colony PCR, 1 pmol of the primer pair of the corresponding gene, 1 μl of 2 mM dNTP, 1 μl of 1 ml / mg BSA, 2.5 μl of Mg _² 10 × buffer, and 1 μl of Taq polymerase were added, Lt; 0 > C for 1 minute and 72 < 0 > C for 2 minutes. The E. coli transformant was cultured in LB medium, centrifuged to collect the cells, and the plasmid DNA was extracted using an alkali extraction method. 100 μl of Sol I, 200 μl of Sol II, and 150 μl of Sol III were added in this order, followed by centrifugation, followed by phenol purification and ethanol precipitation to obtain plasmid DNA. This was dissolved in water, digested with restriction enzymes, and electrophoresed on 1% agarose gel to select the cloned plasmid.

(5) Expression of VP1 antigen protein

In order to express the cloned VP1 gene, the purified plasmid was introduced into BL21 Escherichia coli cells and transformed. In order to express the cloned VP1 gene, BL21 was cultured in SOB medium, and then transferred to 1 L SOB medium for another 3 hours. Then, 1 mM IPTG was added and incubated at 30 ° C. for 5 hours. The transformant Escherichia coli cells were harvested and lysed, and the presence of VP1 protein was confirmed by SDS gel electrophoresis (see FIG. 5).

2. Preparation of immunostrips

To diagnose hepatitis A, an immunostimule containing the VP1 antigen of the expressed HAV was prepared. The immunostrip comprises a sample pad; Conjugation pads; Nitrocellulose membranes; And a hygroscopic pad. The sample pad, the conjugation pad, the membrane, and the hygroscopic pad are fixed on the solid support so as to overlap each other to form one immunostrip as a whole (FIG. 6A).

The conjugation pad of the immunostrip contains gold anti-human IgM conjugated gold nanoparticles.

In the nitrocellulose membrane, the goat anti-mouse IgG antibody was added to the control line, the VP1 antigen protein expressed by the above method was applied to the test line, and the mouse anti-human IgM antibody was immobilized at a constant interval Is fixed.

The sample pad is applied with a diluted test liquid, and the hygroscopic pad located at the uppermost part of the kit serves to induce continuous capillary phenomenon in order to remove unreacted sample or unreacted coloring material from the nitrocellulose membrane do.

3. Diagnostic Kit with Immune Strip

A kit for the diagnosis of HAV infection including the above-prepared immunostrips, sample swabs, diluent (50 mM Tris, 0.01% sodium azide, 0.1% Triton X-100, Respectively.

4. Diagnosis using the diagnostic kit of the present invention

1) When HAV is infected, anti-HAV IgM antibody is usually produced in the infectious agent. The diagnostic kit of the present invention detects an anti-HAV IgM antibody present in a specimen and diagnoses whether it is infected with hepatitis A.

2) First, the specimen is sampled with a swab included in the diagnostic kit, and then diluted with a diluent or the sample itself is applied to the sample pad of the kit. Examples of the specimen include human and animal-derived substances such as whole blood, plasma, serum, tears, saliva, urine, feces, runny nose, body fluids and the like.

3) The immunostrip of the present invention is based on immunochromatography. When a sample is applied to the sample pad, the antibody in the sample moves toward the nitrocellulose membrane by capillary action, and binds to the VP1 antigen on the membrane, Respectively.

Therefore, when the antibody against the corresponding antigen is present in the specimen, both the control line and the test line appear as purple lines. When the corresponding antigen is not present in the specimen, only the control line is developed, and the test line is not developed.

<Result>

1. DNA target location information for subcloning of each gene

Genetic information for HAV-VP1 (NP_740552) used in this study was used to identify the DNA sequence on the NCBI website and to amplify the specific region of the gene specific for each gene. In order to facilitate subcloning after PCR amplification of each gene, restriction enzyme cleavage sequences such as BamH1, HindIII, and EcoR1 were used as primer sequences Lt; / RTI &gt; The information of the gene sequence, the amino acid sequence and the primer sequence are shown in FIG. 2, respectively.

2. PCR amplification at the target position

After confirming the DNA sequence of HAV-VP1 on the NCBI website, PCR amplification was carried out using appropriate primer sets, respectively. As a result, a band of HAV VP1 expected at about 900 bp was confirmed (see left side of FIG. 3). On the other hand, the pET28a vector and the PCR product used for the subcloning were cleaved with restriction enzymes and cloned by ligation with each other. Then, the cloned vector was subjected to restriction enzyme treatment to confirm proper cloning. As shown in the right photograph of FIG. 3, a distinct band was found near 900 bp, confirming that the PCR product was properly inserted into the cloning vector.

3. Ligation and E. coli transformation

The cloned plasmid was transformed into XL-1 blue competent cells. After 24 hours, 6 colonies were selected for each gene and colony PCR was performed. As a result, all of the colonies were positive clones containing an insert for the gene (FIG. 4). Plasmids were also extracted by culturing each colony. The extracted plasmids were transformed into BL21 competent cells and used for protein overexpression experiments.

4. IPTG induction and HAV VP1 antigen expression results

BL21 cells were cultured to over-express HAV-VP1 subcloned into the pET vector. The next day, the cells were transferred to a 200-ml culture container, cultured for another 3 hours, treated with 1 mM IPTG, further incubated for 8 hours at a temperature of 30 ° C. When the transformed E. coli cells were collected and protein overexpression was confirmed, VP1 was confirmed at a size of about 43 kDa. Such overexpressed proteins are of a size that can be inferred from the length of the nucleotide sequence produced at the time of subcloning.

5. Diagnostic results using the diagnostic kit of the present invention

1) In the case of infection with hepatitis A virus, that is, in the presence of IgM antibody against VP1 of HAV in the sample, the anti-VP1 IgM antibody binds to gold nanoparticle-conjugated mouse anti-human IgM on the conjugation pad , It moves laterally by capillary phenomenon, and binds again with the VP1 antigen on the nitrocellulose membrane and develops a purple color on the test line. The gold nanoparticle-conjugated mouse anti-human IgM also binds to a goat anti-mouse IgG antibody and a mouse anti-human IgM antibody on the membrane, respectively, resulting in the presence of a negative control, a test line, A total of three purple bands are shown (Fig. 6B).

2) In the case of not infecting the hepatitis A virus, that is, in the absence of IgM antibody against VP1 of HAV in the sample, there is no gold nanoparticle-conjugated antibody binding to the VP1 antigen on the membrane, No bands appear, and therefore color bands appear only in the control and in the control.

<110> eudipia co., Ltd. <120> DIAGNOSIS KIT FOR HEPATITIS A COMPRISING HAV VP1 ANTIGEN <130> P170360 <160> 4 <170> KoPatentin 3.0 <210> 1 <211> 903 <212> DNA <213> Hepatitis A virus <400> 1 atggtggggg acgactccgg cggtttctct accaccgtga gcacagagca gaatgtaccc 60 gatccccagg tgggcattac cacgatgaaa gatctgaagg gcaaggctaa ccgaggtaag 120 atggacgtca gtggtgtgca agctcccgta ggagccatta ccacgattga agatcctgtt 180 ctcgccaaaa aagtgcctga aacattcccc gaattgaaac ccggggagtc acgccatact 240 tcagaccata tgtccatata taaatttatg ggacggtccc attttctctg cacctttaca 300 ttcaacagta acaataaaga atatacattt ccaattactc tgagctccac ttctaaccca 360 ccgcacggcc tcccctctac cttgagatgg ttcttcaacc tgttccagct gtaccggggg 420 cctctggacc tcacaatcat cattactggc gccacagacg tcgacggaat ggcttggttc 480 acgccggttg gcttggccgt tgatacacct tgggtcgaga aggaatctgc actctccatc 540 gactacaaaa ccgccctggg ggctgtccga ttcaatacca ggaggacagg gaacatacag 600 atccgactcc catggtatag ttatctctac gcagtaagcg gagccctgga cggattggga 660 gataagacag actccacctt tggcctggtc tccattcaga tagctaatta caatcactct 720 gatgaatact tgagcttttc atgttatctg tccgtcaccg agcagtctga gttttacttt 780 cctcgggcac ccctgaatag taatgcaatg ctgagtactg agagtatgat gagccgaatt 840 gctgccggtg acctggagtc ctccgtcgac gatccaagaa gtgaggagga caagcggttt 900 gag 903 <210> 2 <211> 301 <212> PRT <213> Hepatitis A virus <400> 2 Met Val Gly Asp Asp Ser Gly Gly Phe Ser Thr Thr Val Ser Thr Glu   1 5 10 15 Gln Asn Val Pro Asp Pro Gln Val Gly Ile Thr Thr Met Lys Asp Leu              20 25 30 Lys Gly Lys Ala Asn Arg Gly Lys Met Asp Val Ser Gly Val Gln Ala          35 40 45 Pro Val Gly Ala Ile Thr Thr Ile Glu Asp Pro Val Leu Ala Lys Lys      50 55 60 Val Pro Glu Thr Phe Pro Glu Leu Lys Pro Gly Glu Ser Arg His Thr  65 70 75 80 Ser Asp His Met Ser Ile Tyr Lys Phe Met Gly Arg Ser His Phe Leu                  85 90 95 Cys Thr Phe Thr Phe Asn Ser Asn Asn Lys Glu Tyr Thr Phe Pro Ile             100 105 110 Thr Leu Ser Ser Thr Ser Asn Pro Pro His Gly Leu Pro Ser Thr Leu         115 120 125 Arg Trp Phe Phe Asn Leu Phe Gln Leu Tyr Arg Gly Pro Leu Asp Leu     130 135 140 Thr Ile Ile Thr Gly Ala Thr Asp Val Asp Gly Met Ala Trp Phe 145 150 155 160 Thr Pro Val Gly Leu Ala Val Asp Thr Pro Trp Val Glu Lys Glu Ser                 165 170 175 Ala Leu Ser Ile Asp Tyr Lys Thr Ala Leu Gly Ala Val Arg Phe Asn             180 185 190 Thr Arg Arg Thr Gly Asn Ile Gln Ile Arg Leu Pro Trp Tyr Ser Tyr         195 200 205 Leu Tyr Ala Val Ser Gly Ala Leu Asp Gly Leu Gly Asp Lys Thr Asp     210 215 220 Ser Thr Phe Gly Leu Val Ser Ile Gln Ile Ala Asn Tyr Asn His Ser 225 230 235 240 Asp Glu Tyr Leu Ser Phe Ser Cys Tyr Leu Ser Val Thr Glu Gln Ser                 245 250 255 Glu Phe Tyr Phe Pro Arg Ala Pro Leu Asn Ser Asn Ala Met Leu Ser             260 265 270 Thr Glu Ser Met Met Ser Arg Ile Ala Ala Gly Asp Leu Glu Ser Ser         275 280 285 Val Asp Asp Pro Arg Ser Glu Glu Asp Lys Arg Phe Glu     290 295 300 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> HAV-VP1-EcoR-S1 <400> 3 gaattcggcg gtttctctac caccgtg 27 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> HAV-VP1-Hind-AS1 <400> 4 aagcttgctt ctcctcactt cttgga 26

Claims (5)

A composition for the diagnosis of hepatitis A comprising the VP1 antigen of HAV (Hepatitis A virus). 2. The diagnostic composition according to claim 1, wherein the VP1 antigen is a protein of SEQ ID NO: 2 or an immunologically equivalent protein thereof. A hepatitis A diagnostic kit comprising the diagnostic composition of claim 1 or 2. The diagnostic kit according to claim 3, wherein the kit is one selected from the group consisting of an RT-PCR kit, an immunostrip, a biochip, and an ELISA diagnostic kit. The diagnostic kit according to claim 3, wherein the test strip is an immunostrip.

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