CN104374915B - For detecting cAg fragment and the application thereof of hepatitis C virus - Google Patents

Abstract

The present invention provides the cAg fragment for detecting hepatitis C virus and application thereof. Do you use Abbott Laboratories Architect? the antigen levels of the different HCV genotype serum sample of HCV-cAg quantification kit detection, the relatively sensitivity of detection different genotype, the sample of the specific gene type of analyzing influence detection sensitivity, searching out the pleomorphism site of the cAg affecting test kit detection sensitivity, is its aminoacid sequence such as SEQ? ID? shown in NO.1��2. The amino acid fragment of this cAg pleomorphism site can be used as the molecule marker of HCV-cAg detection for detecting hepatitis C virus 3b genotype, and the existence of this molecule marker can indicate existing antigen quantify detection kit sensitivity decrease when detecting hepatitis C virus 3b genotype sample, contribute to standardization that cAg detects and stdn, can be used for preparation detection HCV? the test kit of 3b genotype.

Description

Core antigen fragment for detecting hepatitis C virus and application thereof

technical field

The invention relates to the field of virus detection, in particular to a core antigen fragment for detecting hepatitis C virus and its application.

Background technique

Chronic hepatitis C (CHC) caused by persistent infection of hepatitis C virus (HCV) has been a global public health problem. The onset of CHC is hidden and easily ignored by patients. It can lead to liver cirrhosis and even hepatocellular carcinoma and liver failure, posing a serious threat to human health. Therefore, the screening and early treatment of HCV infection is particularly important. The common hepatitis C genotypes in my country mainly include 1b, 2a, 3a, 3b and 6a genotypes.

Anti-HCV detection is the most widely used screening test for HCV infection. It is easy to operate, short in time, and low in cost. However, its disadvantage is that it has a long window period and cannot distinguish whether it is an active infection or the virus has been cleared. It is not suitable for immunodeficiency population; HCV RNA is the direct evidence of viral infection, and quantitative detection of HCV RNA is also a key indicator for monitoring the efficacy of CHC patients during antiviral treatment. Operations are prone to cross-contamination and aerosol pollution, and are not suitable for small laboratories. Compared with anti-HCV and RNA detection, HCV core antigen detection combines the advantages of the above two detections, namely, simple operation, short time consumption, easy automation, high detection precision, and greatly reduced possibility of contamination. At the same time, as a As a direct marker of viral replication, anti-HCV testing should not be selected when screening for HCV infection in immunocompromised or deficient groups such as AIDS patients, kidney disease patients receiving long-term dialysis, organ transplant patients or patients with congenital immune deficiency, Optional core antigen testing. There are also studies suggesting that early monitoring of HCV antigen levels in the treatment of CHC with interferon combined with ribavirin can be used to predict SVR.

Although the detection of HCV core antigen has many advantages, the content of HCV antigen in serum is very low, so the requirements for detection technology are very high. In recent years, with the development and maturity of immunological detection technologies such as chemiluminescence, sensitive, quantitative and suitable HCV antigen detection reagents for automatic detection have been successfully developed. The internationally mainstream Abbott Architect HCV Ag kit uses c11-9 and c11-14 Two monoclonal antibodies are used as detection antibodies, and the corresponding epitopes are HCV core antigen amino acid 21-40 fragment (DVKFPGGGQIVGGVYLLPRR) and core antigen amino acid 41-50 fragment (GPRLGVRATR). Abbott Architect HCVAg detection kit can be used for early diagnosis of HCV infection, and can also be used to monitor the virological response of CHC antiviral therapy. It has been widely used in the field of HCV diagnosis and treatment, and is currently the most widely used HCV antigen detection kit in the world . However, in the practice of clinical testing, some samples of specific genotypes appeared. Although the concentration of HCV RNA was high, indicating that the virus was in the active replication phase, the result of quantification of HCV core antigen using this kit was "negative", which shows that the current The HCV detection kit still has the problem of false negatives for some samples, that is, its detection sensitivity to some positive HCV samples is significantly reduced, but no report has pointed out the reasons for the reduction in detection sensitivity of these samples.

The correct diagnosis of HCV in the sample has important guiding significance for the early intervention and treatment of patients with early hepatitis C virus infection. If the detection sensitivity of some specimens in the existing kits can be overcome, it will help the early detection of HCV. diagnosis and treatment.

Contents of the invention

The first object of the present invention is to provide core antigen fragments for detecting hepatitis C virus.

The second object of the present invention is to provide the application of the core antigen fragment.

The present invention uses ArchitectHCVAg reagent to detect the HCV antigen in the serum of CHC patients in China, and compares the sensitivity of detecting different genotypes by detecting the antigen levels of serum samples of different HCV genotypes, and further detects the core antigen of samples of specific genotypes that affect the detection sensitivity The amino acid sequence 21-50 fragment (DVKFPGGGQIVGGVYLLPRRGPRLGVRATR), the applicant found that there are multiple amino acid polymorphism sites in the amino acid 21-50 region of the core antigen, and D21N, L36V and T49V coexist or D21N, L36V, P42L and T49V coexist. Affect the detection performance of the Abbott ArchitectHCVAg kit, resulting in a decrease in its detection sensitivity. The above two amino acid polymorphisms all appear in the 3b genotype of hepatitis C virus.

Therefore, the present invention provides a core antigen fragment for detecting hepatitis C virus, the amino acid sequence of which contains the amino acid fragment shown in SEQ ID NO.1 or SEQ ID NO.2. which is:

SEQ ID NO: 1: N VKFPGGGQIVGGVY V LPRRG L RLGVRA V R

SEQ ID NO: 2: N VKFPGGGQIVGGVY V LPRRGPRLGVRA V R The above fragments with amino acid polymorphisms can be used as molecular markers for detecting hepatitis C virus, especially for detecting 3b genotype hepatitis C virus. For the convenience of expression, the amino acid polymorphism "pattern 1" described in this application is the amino acid fragment shown in SEQ ID NO.1, and "pattern 2" is the amino acid fragment shown in SEQ ID NO.2.

The nucleotide sequences of the genes encoding the above two core antigen fragments are respectively shown in SEQ ID NO.5 or SEQ ID NO.6.

The application of the expression vector containing the gene shown in SEQ ID NO.5 or SEQ ID NO.6 in the preparation of the hepatitis C virus detection kit also belongs to the protection scope of the present invention.

The present invention also provides the application of the above-mentioned core antigen fragment in the preparation of a 3b genotype hepatitis C virus diagnostic kit. When the amino acid sequence of the detected antigen fragment contains the sequence shown in SEQ ID NO.1 or SEQ ID NO.2, the detected virus is of HCV3b genotype.

The present invention provides the application of the above-mentioned core antigen fragment or its coding gene in the preparation of 3b genotype hepatitis C virus antibody diagnostic reagent.

The application of the above-mentioned core antigen fragments in the preparation of 3b genotype hepatitis C vaccine also belongs to the protection scope of the present invention.

The present invention also provides a monoclonal antibody whose corresponding antigenic epitope contains the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2.

Hybridoma cell lines secreting the above-mentioned monoclonal antibodies also belong to the protection scope of the present invention.

The present invention provides the application of the above-mentioned core antigen fragment in preparing a kit for identifying the hepatitis C virus 3b genotype and other genotypes, when the amino acid sequence of the detected antigen fragment contains the sequence shown in SEQ ID NO.1 or SEQ ID NO.2 When, the detected virus is HCV3b genotype.

The invention provides a kit for detecting hepatitis C virus containing the core antigen fragment of the invention.

A kit for preparing and detecting hepatitis C virus containing any primer pair capable of amplifying the gene described in SEQ ID NO.5 or SEQ ID NO.6 also belongs to the protection scope of the present invention.

Preferably, the above kit for detecting hepatitis C virus is a kit for detecting 3b genotype hepatitis C virus.

The present invention has discovered that the kit for detecting hepatitis C virus in the prior art detects the amino acid 21-50 region of the core antigen of HCV, and there are many amino acid polymorphism sites, which cause its detection sensitivity to decrease, while the above two amino acid polymorphisms Both sexes are present in the 3b genotype of the hepatitis C virus. This means that the detection sensitivity of the prior art to the 3b genotype of hepatitis C virus is not high, and false negative results are prone to occur. The present invention creatively found that the amino acid fragment of the polymorphic site of the core antigen can be used as a molecular marker for the detection of HCV core antigen to detect the HCV 3b genotype, and the presence of the molecular marker can indicate the existing quantitative antigen The detection sensitivity of the detection reagent is reduced, which contributes to the standardization and standardization of HCV core antigen detection, and can be used to prepare a test kit for detecting HCV3b genotype, which helps to make up for the lack of sensitivity of the existing kits to detect HCV, and further improves the existing technology. The detection sensitivity and accuracy of the kit for detecting hepatitis C virus make it more broad-spectrum and effective.

Description of drawings

Figure 1 is a comparison of the amino acid sequences of the serum HCV core region of patients with different genotypes of chronic hepatitis C (from the 21st to the 50th amino acid of the core antigen), and the numbers in the leftmost column are the sample numbers and their genotypes.

Figure 2 is the result of western blot confirmation of FLAG-HCV core protein polypeptide (including amino acid polymorphism sites) expressed in mammalian cell HEK293T. ** is the 3b genotype sample containing the amino acid polymorphism "pattern 1"; * is the 3b genotype sample containing the amino acid polymorphism "pattern 2".

Figure 3 is a comparison of the results of Abbott Architect HCV antigen quantitative detection of HEK293T expression products of each genotype. ** is the 3b genotype sample containing the amino acid polymorphism "pattern 1"; * is the 3b genotype sample containing the amino acid polymorphism "pattern 2".

detailed description

The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, any modifications or substitutions made to the methods, steps or conditions of the present invention fall within the scope of the present invention.

Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

Example 1 Comparison of Core Antigen Levels in Different HCV Genotype Samples

1. Case selection

From March to June 2011, serum was collected from 203 CHC patients, including 130 males and 73 females, aged 24-76 years, with an average of (37.3±8.6) years old. HCV genotypes were detected using VersantHCV Genotype2.0 (LiPA, Siemens AG, Marburg, Germany), of which 41 cases were genotype 1b, 43 cases were 2a, 31 cases were 3a, 43 cases were 3b, and 45 cases were 6a, which basically covered Common genotypes in China. HCV RNA was quantified using the Roche Taqman HCV RNA Kit, with a minimum detection limit of 15 IU/mL. Patients with different HCV genotypes were comparable in terms of age, gender and HCV viral load.

2. Method

Abbott Architecti2000 automatic chemiluminescence instrument was used for quantitative detection of HCV antigen by Abbott Architect HCVAg core antigen quantitative detection kit. The linear range of quantitative detection is 3.00fmol/L-20000fmol/L, and the lowest detection limit is 3.00fmol/L. One-way analysis of variance and LSD method were used to compare the overall mean and multiple comparisons of the mean of each group, and P<0.05 was considered significant in the difference.

3. Results

The quantitative results of HCV antigen in different genotype patient groups are shown in Table 1. The results showed that the 3b genotype had lower antigen levels than the other genotypes.

Table 1 Comparison of serum HCV core antigen levels in patients with different genotypes

Embodiment 2 compares the sensitivity of Abbott Architect HCVAg Quantitative Kit to detect different genotype HCV samples

1. Sample selection Select 1b, 2a, 3a, 3a and 6a genotypes of high viral load serum each.

2. Experimental method Use the Abbott Realtime HCV RNA Quantitative Kit to detect the viral load of each genotype sample, and then dilute the serum of each genotype so that the final concentration of HCV RNA is 10000, 7500, 5000, 1000, 750, 500, 250 and 100IU/ mL. Diluted samples with different concentrations were tested 20 times using Abbott Architect HCVAg quantitative reagent. Use the probability analysis method (Probit method) to calculate the corresponding minimum HCV RNA concentration when 95% of the samples are "positive" by Abbott Architect antigen quantitative detection, and this concentration is regarded as the sensitivity of the Architect HCVAg quantitative kit, that is, the "lowest detection limit" .

3. Experimental results

The sensitivity of serum Abbott Architect HCV Ag to detect 3b genotype samples is much lower than that of other genotypes (see Table 2). Therefore, it is necessary to determine the amino acid sequence of the 3b HCV core antigen to explore whether there are polymorphic sites that affect quantitative detection.

Table 2 Comparison of detection sensitivity of ArchitectHCVAg quantitative kit in patients with different genotypes

Example 3 Alignment of Amino Acid Sequences of Core Regions of Different Genotypes of HCV Strains

1. Materials and methods

(1) Serum HCV RNA extraction

HCV RNA in 140 μl serum was extracted using the QIAamp Viral RNA Extraction Kit from Kaiser, Germany.

(2) HCV core region reverse transcription and PCR amplification (RT-PCR)

HCV RNA was reverse-transcribed using AMV reverse transcriptase from Promega, USA. The total volume of RT reaction system for 1 person is 10 μl, including 6 μl HCV RNA, 1× AMV reaction buffer, 1 mM each dNTP, 3 μM RT primer Core410R and 6 U AMV reverse transcriptase. The RT reaction conditions are: 42°C, 60min. Add 40 μl PCR reaction solution to 10 μl 5'-UTR reverse transcription system, so that the total volume of the PCR reaction system is 50 μl, including 2.5U PlatinumTaq DNA polymerase from Invitrogen Company, 1×PCR buffer, 1.5mMMg ²⁺ , 200μM each dNTP, 0.6 μM primer Core406F. PCR conditions are: 95°C, 5min for DNA denaturation, then 95°C, 30sec→48°C, 30sec→72°C, 1min, repeat 40 cycles; 72°C, 10min, cool to 4°C. See Table 3 for the sequences of Core410R and Core406F primers and their positions in the HCV genome.

Table 3 is used for the primer of amplification HCV core region

(3) The amino acid numbering principle and comparison method of HCV core region

The HCV nucleic acid and amino acid sequence numbering principles are based on the HCV strain H77 (GenBank registration number AF009606) as the reference virus strain, following the international general numbering rules published in Hepatology in 2006. Use BioEdit7.0 software for RNA to amino acid translation and sequence alignment.

2. Results Comparison of amino acids in the core region of different genotypes of HCV showed that, compared with the 21-50 fragment (DVKFPGGGQIVGGVYLLPRRGPRLGVRATR) of other subtypes, the 3b subtype had multiple amino acid polymorphism sites in the core region aa21 to aa50 (See Figure 1), there are 2 polymorphism patterns, the specific sequences are shown in SEQIDNO.1 and SEQIDNO.2:

Polymorphism pattern 1: core antigen: D21N+L36V+P42L+T49V

Polymorphism pattern 2: core antigen: D21N+L36V+T49V

The gene sequences encoding the core antigen fragments of the above two polymorphisms are respectively shown in SEQ ID NO.5 and SEQ ID NO.6.

Example 4 In vitro experiments verify the impact of amino acid polymorphism sites in the HCV core antigen region on the detection sensitivity of the Abbott Architect HCVAg kit

1. Materials and methods

From the 203 parts of patient serum in embodiment 1, select 6 parts of representative samples of HCV genotype, are respectively: 01037 (1b type), 01049 (2a type), 25026 (3a type), 07001 (3b type, polymorphism Pattern 1), 07035 (type 3b, polymorphism pattern 2) and 59526 (6a), extract viral RNA, RT-PCR, see Example 3 for the reaction conditions. The 411bp cDNA sequence (nt342-nt752) of the obtained HCV core region was inserted into the pCAG mammalian expression vector. The pCAG plasmid has a FLAG tag to facilitate the identification and purification of the expression product; the insertion site is an EcoRI restriction site. The expression plasmid was transfected into HEK293T cells by calcium phosphate co-precipitation method. The specific operation steps were: subculture HEK293T 1 day before transfection, and transfect when the cells were 80%-85% full; replace the medium before transfection For DMEM (containing 5% serum), the calcium phosphate-pCAG-HCVcore plasmid complex was added to the surface of HEK293T monolayer cells. After 6 hours of transfection, replace with DMEM (containing 10% serum) and culture for 8 hours, and then replace the medium with expression medium (100 μg BSA/mLDMEM). Cells were harvested 48 h after transfection. After ultrasonically lysing cells, centrifuge to remove cell fragments, use the bicinchoninic acid method (Pierce, USA) to quantify the total protein in each tube, and adjust the total protein content of each portion to the same concentration according to the results of total protein quantification. Take 50 μL of each total protein for sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot to identify the target protein. The antibody used was mouse anti-human M2-Flag monoclonal antibody (Sigma, Germany).

2. Results

(1) The results of western blot detection of mammalian cell expression products are shown in Figure 2. The expression of core protein in each genotype was basically at the same concentration level. The molecular weight of the expressed protein is about 16.46kD. The Abbott ArchitectHCVAg kit was used to quantitatively detect the core protein in each expression product. The quantitative results (see Figure 3) showed that, compared with other genotypes, the 3b type recombinant core protein had two amino acid polymorphism patterns, which all resulted in higher The quantitative detection result is low (only 30%-40% of the core protein quantitative value detected by other genotypes).

Example 5 Application of samples from patients with chronic hepatitis C (before antiviral treatment) to verify the impact of core antigen amino acid polymorphisms on the Abbott Architect HCVAg quantitative kit

1. Materials and methods

One case of type 3b serum samples with polymorphism pattern 1 and polymorphism pattern 2 in the core antigen region were selected, and the sample numbers were 07001 and 07035 respectively; at the same time, one case of other representative genotype (1b and 2a) samples were selected , there is no polymorphic site in the core antigen region, and the sample numbers are 01037 and 01049, respectively. Each sample was detected 5 times with Abbott Architect HCVAg reagent, and the mean value and standard deviation (SD) were taken. The HCV RNA load of all samples is close (5.60-5.80log10IU/mL), so as to visually compare the detection sensitivity of Abbott ArchitectHCV Ag reagent to different samples.

2. Results

Compared with 1b and 2a genotype samples without core region polymorphism patterns, ArchitectHCVAg Quantitative Kit detected 3b samples with 2 kinds of amino acid polymorphism patterns, and the detection results were more than 10 times lower (>1.00log10fmol/L ), see Table 4. Thus, it was confirmed that polymorphism pattern 1 and polymorphism pattern 2 can lead to a decrease in the detection sensitivity of the reagent. This finding indicated that the Abbott Architect HCV Core Antigen Quantitative Reagent was affected by multiple amino acid polymorphism sites in 3b genotype samples, resulting in quantitative results that were only about 10% of the expected value.

Table 4 Antigen quantitative results of different genotype samples by ArchitectHCVAg quantitative kit

Example 6 In patients with chronic hepatitis C (during antiviral therapy), the influence of core antigen amino acid polymorphisms on the Abbott Architect HCVAg quantitative detection kit was verified

1. Case selection

Among the 203 patients with chronic hepatitis C in Example 1, 11 patients received pegylated interferon combined with ribavirin antiviral therapy and retained a sufficient amount of HCV at the end of treatment 0w, 4w, 12w and the end of treatment. Serum after 24w. Among the 11 patients, 7 were genotype 1b, 2 were genotype 2a, and 2 were genotype 3b (see Table 5 for detailed data).

Among the 11 patients, 7 patients achieved rapid virological response (rapid virological response, RVR), that is, the serum HCV RNA was "negative" after 4 weeks of treatment; the remaining 4 patients did not achieve RVR. During the continuation of antiviral treatment, these 4 patients all obtained early virological response (early virological response, EVR), that is, the detection of serum HCV RNA was "negative" after 12 weeks of treatment; all patients obtained sustained virological response after the end of treatment. Response (sustained virological response, SVR), that is, half a year after the end of treatment, serum HCV RNA is "negative".

2. Method

Abbott Architect HCVAg Core Antigen Quantitative Kit was used to detect the core antigen levels in serum of 11 patients at 0w, 4w, 12w and 24w after treatment.

3. Experimental results

Among the serial serum test results of 11 patients, the test results of HCV RNA and core antigen were consistent in 10 cases. However, there was one patient with genotype 3b (patient number: 49077) whose RNA was positive at 2.10×10 ³ IU/mL at 4 weeks of treatment, but the quantitative detection result of HCV core antigen was negative (<3.00 fmol/L). See the table for details 5. The results showed that during the antiviral treatment of chronic hepatitis C patients, the virological response monitoring at the key time point 4w, the detection sensitivity of Abbott Architect HCVAg kit to detect the core antigen in patients with genotype 3b is insufficient, which may lead to the misjudgment of non-RVR patients as RVR , which may lead to insufficient treatment courses and cause viral rebound or recurrence, which will increase unnecessary economic and mental burdens on patients.

Table 511 Cases of Chronic Hepatitis C Patients Pegylated Interferon Combined with Ribavirin Therapy Comparison of HCV RNA and Core Antigen Quantitative Detection Results at Key Time Points

*HCV RNA, log10IU/mL; **HCV core antigen, log10fmol/L

Although, the above has described the present invention in detail with general description, specific implementation and test, but on the basis of the present invention, some modifications or improvements can be made to it, which will be obvious to those skilled in the art of. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (9)

1., for detecting the encoding gene of the cAg fragment of hepatitis C virus 3b genotype, its nucleotide sequence is respectively as shown in SEQIDNO.5 or SEQIDNO.6.

2. the cAg fragment of aminoacid sequence as shown in SEQIDNO.1 or SEQIDNO.2 is in the application prepared in hepatitis C virus 3b gene type diagnostic test kit.

3. gene according to claim 1 is in the application prepared in hepatitis C virus 3b gene type diagnostic test kit.

4. the expression vector containing gene described in claim 1 is in the application prepared in hepatitis C virus 3b gene type diagnostic test kit.

5. the application of the cAg fragment of aminoacid sequence as shown in SEQIDNO.1 or SEQIDNO.2 in preparation 3b genotype antibody of HCV diagnostic reagent.

6. the application of the cAg fragment of aminoacid sequence as shown in SEQIDNO.1 or SEQIDNO.2 in preparation 3b genotype hcv vaccine.

7. a hepatitis C virus 3b genotype monoclonal antibody, it is characterised in that, the aminoacid sequence of the epitope of its correspondence is as shown in SEQIDNO.1 or SEQIDNO.2.

8. secrete the hybridoma cell strain of monoclonal antibody described in claim 7.

9. contain the test kit of the detection hepatitis C virus 3b genotype of the primer pair of gene described in amplification claim 1.