CN1188427C - Recombinant chimeric peptide antigen detecting HCMV antibody and production method thereof - Google Patents

Abstract

The present invention discloses a recombinant chimeric peptide antigen for detecting an HCMV antibody and a producing method thereof. The antigen prepared from target protein only containing a serial determinant fragment of HCMVgp52CTPpp150CTP has strong specificity and high sensitivity. The producing method is carried out in a secretion type yeast expression system to ensure that the antigen is a chimeric peptide only containing target protein.

Description

Recombinant chimeric peptide antigen for detecting HCMV antibody and production method thereof

Technical field:

The invention discloses a chimeric peptide antigen for detecting HCMV antibody and a production method thereof. The object type is a multi-determinant antigen for detecting HCMV antibody and its expression production method constructed by recombinant PCR technology in culture.

Background technique:

Human cytomegalovirus (HCMV) is a widespread human pathogen that is transmitted naturally through saliva, urine, or lactation, as well as through sexual contact, blood transfusion, and organ transplantation [Greijer A E, Crommert JG, Stevens SC, et al. Moleculae fine-specificity analysis of antibody responses to human cytomegalovirus and design of novelsynthetic-peptide-base serodiagnostic assays. J Clin Microbiol, 1999; 37(1): 179-188.]. It mainly causes subclinical infection in humans, but for immunosuppressed individuals and patients receiving tumor treatment, HCMV infection can induce many diseases, leading to serious diseases and even life-threatening. In addition, HCMV is also one of the important pathogenic factors affecting eugenics. Among fetuses and newborns infected with primary HCMV during pregnancy, 80% can lead to mental retardation, deformity and death [Middeldorp JM. Jongsma J, Haar AT, et al .Detection of immunoglobulin M and G antibodies against cytomegalovirus early andlate antigens by enzyme-linked immunosorbent assay.J Clin.Microbiol, 1984;20(4):763-771.]. Therefore, rapid and accurate diagnosis of viral infection is very important for the diagnosis and treatment of HCMV-related diseases, especially for the detection of pregnant women and blood donors.

At present, the serological method commonly used for antibody detection is enzyme-linked immunosorbent assay (ELISA). However, when a serum sample is tested with different ELISA kits, different results often appear [WeberB, Prosser F, Munkwitz A, et al.Serological diagnosis of cytomegalovirus infection: comparison of eight enzyme immunoassays for the detection of HCMV-specific IgMantibody . Clin Diag Virol, 1994;2:245-259.]. This is mainly because the antigens used in the ELISA kits currently used in the world are all derived from the cell culture of the virus, that is, the whole virus. Studies have found that only a limited number of HCMV proteins can react with positive sera in different stages of clinical infection, which is necessary for the serological diagnosis of HCMV. Since many HCMV antigen components have strong homology with corresponding components in other herpes viruses, and the purified whole virus antigen often contains host cell antigen components, the specificity, sensitivity and stability of the results vary greatly[ Chee MS, Bankier S, et al. Curr. Top. Mirobiol. Immunol., 1990; 154: 125-169.]. Therefore, the use of HCMV proteins or polypeptides with strong immunogenicity and no homology with other viruses as antigens to replace whole virus antigens is an effective way to improve the sensitivity and specificity of HCMV infection diagnosis.

Serological diagnosis requires that the antigenic determinant of HCMV protein can be recognized by IgM and IgG that appear at different stages of HCMV infection. Therefore, finding antigenic peptides with strong immunogenicity has always been the goal of researchers. Greijer et al. used whole virus extracts for immunoblot analysis to better identify immunogenic peptides in HCMV. It was found that gp52 was the first HCMV protein recognized for the IgM and IgG responses in most patients. In addition, Ripalti [Ripalti A, Landini MP, Mocarski ES, et al. Identification and preliminary use of recombinant Lamda gtll fusion proteins inhuman cytomegalovirus diagnosis. J Gen Virol, 1989; 70: 1247-1251.] using HCMV-positive human serum from HCMV Reactive clones were isolated from the λgtll expression library of DNA. Among the resulting recombinant clones, one is a partial peptide of the important HCMV structural phosphoprotein pp150, and the other is an important non-structural protein gp52. Spaete [Spaete RR, Gehrz R, Landini MP, et al. Humancytomegalovirus structural proteins. J Gen Virol, 1994; 75: 3287-3308.] confirmed that in viral particles, the inner membrane pp150 is the most immunogenic protein. Using the immunoblotting method to detect their binding specificity, it was found that in the sera of patients with acute HCMV infection, antibodies to the DNA-binding protein gp52 showed high titers, while the high titers of antibodies to the structural phosphoprotein pp150 were ubiquitous In sub-health HCMV seropositive. The experimental results showed that pp150 and gp52 are important protein antigens for detecting HCMV infection.

A single antigenic determinant of a single protein can only be recognized by some specific antibodies, and cannot replace the application of the whole virus antigen in serological diagnosis. Therefore, some people consider using different antigenic determinants in series to detect specific antibodies. Greijer et al. recombined partial peptides at the carboxy-terminus of pp150, ie 1011-1048 amino acids, and 266-293 amino acid residues at the carboxy-terminus of gp52, and used ELISA to detect IgM with a sensitivity as high as 96.4%. Ruan Qiang et al [Ruan Qiang, Ripalti A, Landini MP, et al. The role of recombinant human cytomegalovirus ppUL32 protein in diagnosis. Chinese Journal of Microbiology and Immunology, 1995; 15(2): 143-144.] The two gene products of nucleotides 1783-1842 and nucleotides 3072-3144 of pp150 protein were recombined in the same vector. Compared with a single gene, the fusion protein expressed by its clone has stronger reactivity with HCMV positive sera, and the recognition rates with HCMV IgM and IgG positive sera are 100% and 68.42%, respectively.

In recent years, gene recombination technology has provided a broad field for obtaining HCMV diagnostic antigen. So far, gene recombination technology has used four expression systems: prokaryotic E. coli expression system, eukaryotic yeast expression system, insect cell virus expression system and Chinese hamster ovary cell (Chinese hamster ovary cell, CHO) expression system. However, the application of HCMV virus protein expression only stays in the prokaryotic Escherichia coli expression system. The main disadvantage of using prokaryotic E. coli to produce HCMV antigens in serological detection is the high level of false positive reactions, because there are often antibodies against E. coli proteins in human serum, and the expression products of this system are all fusion proteins. That is, in addition to the target protein, it also contains E. coli protein.

Invention content:

The object of the present invention is to provide a chimeric peptide antigen for detecting HCMV antibody with strong specificity and high sensitivity, which is constructed in series with different antigenic determinants, contains only target protein, and its recombinant PCR process and expression production method.

The present invention is realized by the following technical scheme: the recombinant chimeric peptide antigen for detecting HCMV antibodies is composed of the DNA sequence of 232 amino acids at the C-terminal of gp52 and 43 amino acids at the C-terminal of pp150, which are concatenated into gp52CTPpp150CTP gene fragments by recombinant PCR technology expression plasmid, and express the target protein in yeast as an antigen.

The recombinant chimeric peptide of the present invention is produced by the following method: respectively amplifying and recombining the gp52CTP and pp150CTP fragments to obtain the gp52CTPpp150CTP recombinant fragment; the pPIC9K vector and the PCR amplification product are digested with EcoRI and NotI and ligated in vitro Transformation and sequencing to identify recombinants; use Sac I enzyme to linearize the recombinant expression plasmid and introduce it into yeast GS118 to select positive clones, screen multiple copies of transformed bacteria, select positive clones for induction culture, secrete proteins, and obtain purified recombinant proteins after treatment .

The detection HCMV antibody chimeric peptide antigen of the present invention is composed of different antigenic determinant components, and only contains the target protein antigen, so the specificity is strong and the sensitivity is high.

The chimeric peptide antigen expression method of the present invention uses the target peptide gene sequences on HCMV gp52CTP and pp150CTP for recombinant PCR, and cultures the constructs in yeast to produce them, thus ensuring that the product is a purified chimeric protein containing only the target protein. peptide antigen.

Detailed ways:

Constructing a plasmid containing only the target peptide sequence on HCMV gp52CTP and pp150CTP, and expressing an antigen containing only the target peptide to recognize IgM and IgG at different stages of HCMV infection will greatly improve the recognition rate. It avoids the disadvantage of high-level false positive reaction in serological detection due to the expression product containing E. coli protein when HCMV antigen is produced in the prokaryotic E. coli expression system, and also avoids the detection of whole virus antigen Cross-reactivity of HCMV antibodies with other herpesviruses. The specific composition method is as follows:

1. Preparations before reorganization

According to the DNA sequence of the target fragment, namely the cDNA sequence of the target peptide on HCMV gp52CTP and pp150CTP and the restriction site on the plasmid, two pairs of PCR primers (p1, p2), (p3, p4) were designed. p1 and p2 are used to amplify the 606th to 1299th nucleotides of gp52, p3 and p4 are used to amplify the 3018th to 3144th nucleotides of pp150; primers p1 and p4 have EcoRI and NotI restriction sites respectively, and primer p2 It is complementary to the sequence of p3, and corresponds to the 3' end of the gp52 target gene and the 5' end sequence of the pp150 target gene.

2. PCR recombination of antigen gene fragments

The first round of PCR amplification: using the virus culture supernatant as a template, primers p1 and p2 amplify the gp52CTP fragment, and primers p3 and p4 amplify the pp150CTP fragment.

The second round of PCR amplification: the gp52CTP and pp150CTP fragments obtained in the first round of PCR amplification were used as templates, and primers p1 and p4 were added to amplify the gp52CTPpp150CTP fragment. In this way, the tandem recombinant fragment of the target gene can be obtained: gp52CTPpp150CTP.

3. Insert the recombinant fragment into the expression plasmid

The pPIC9K vector and the PCR amplification product were digested with EcoRI and NotI, and after purification, the product was ligated with _T4 ligase at 16°C. The ligated product was transformed into Escherichia coli DH5α, and spread on the LB plate containing ampicillin (AMP). Cultivate overnight at 37°C, and the colonies evenly distributed on the plate are observed the next day, which is the screened transformant. Transformants were picked, and plasmid DNA was extracted for sequencing identification.

The sequencing results showed that the target fragment was correctly inserted into the expression plasmid pPIC9K.

4. Expression of antigenic protein in yeast

The PCR recombinant expression plasmid was linearized with Sac I enzyme, introduced into yeast GS118 by electroporation technology, and positive clones were selected by MD plate. Multiple copies of transformed bacteria were screened on G418-YPD plates with increasing concentrations (G418 concentration was 0.5-4.0 mg/ml). The selected positive clones were cultured with YPD solution containing 0.5%-2% methanol to induce their protein secretion, and the supernatant was harvested after continuous induction for 4 days, and the purified recombinant protein was obtained after 75% ammonium sulfate precipitation and gel column chromatography.

5. Western-blot verification of recombinant protein:

In order to detect the degree of cross-reaction between it and HCMV antiserum, it is suggested that the target gene is expressed and has antigenicity, and the following test is carried out. 6 rabbits were immunized with HCMV virus culture supernatant, 6 control rabbits and 50 normal human sera were tested for their cross-reactivity with expressed antigens by Wester-blot. The experimental results are as follows: serum sample total number of cases positive cases Negative cases positive detection rate Negative detection rate immune rabbit 6 6 0 100% 0 control rabbit 6 0 6 0 100% normal person 50 0 50 0 100%

The results show that: the positive sera obtained from 6 rabbits immunized with HCMV virus culture supernatant all produce positive reactions with expressed antigens, and 6 control rabbits and 50 routine normal human sera all produce negative reactions with expressed antigens (i.e. negative and positive detection The average rate reached 100%), suggesting that the target gene was expressed, and the chimeric peptide antigen had cross-reactivity with the whole virus antigen, and had strong specificity, which is expected to replace the whole virus antigen to detect HCMV antibody.

Claims (3)

1, a kind of recombinant chimeric peptide antigen that detects HCMV antibody is characterized in that: described chimeric peptide antigen HCMVgp52CTPpp150CTP contains terminal 232 amino acid of HCMVgp52C and terminal 43 amino acid of pp150C successively from the N-end to the C-end.

2, a kind of method that produces the recombinant chimeric peptide antigen of detection HCMV antibody as claimed in claim 1, its dna recombinant expression comprises that HCMV gp52CTP and pp150CTP go up the recombinant PCR of purpose peptide fragment gene sequence, gene fragment inserts carrier and target protein is expressed, it is characterized in that: the purpose peptide fragment gene sequence on the described gp52CTP is terminal 232 the amino acid whose dna sequence dnas of coding gp52C, the last purpose peptide of pp150CTP fragment gene sequence is terminal 43 the amino acid whose dna sequence dnas of coding pp150C, and described target protein is expressed and carried out in yeast.

3, a kind of generation as claimed in claim 2 detects the method for the recombinant chimeric peptide antigen of HCMV antibody, it is characterized in that: described gene recombination process is made up of following steps: the PCR primer that 1. is designed for amplification gp52 the 606th～1299 Nucleotide is to (p1, p2), be used to increase the PCR primer of pp150 the 3018th～3144 Nucleotide to (p3, p4), primer p1 and p4 have the restriction enzyme site of EcoRI and NotI respectively, primer p2 and p3 order are complementary, and jointly corresponding to gp52 above-mentioned segmental 3 ' the terminal and above-mentioned segmental 5 ' end sequence of pp150; 2. be template with the virus-culturing fluid supernatant earlier, with primer p1, p2 amplification gp52CTP fragment, with primer p3, p4 amplification pp150CTP fragment is a template with gp52CTP and the pp150CTP fragment that the previous round pcr amplification obtains again, adds primer p1 and p4, amplification gp52CTPpp150CTP fragment obtains placed in-line goal gene recombinant fragment: gp52CTPpp150CTP; 3. with pPIC9K carrier and pcr amplification product through EcoRI and NotI double digestion, product purification is after T ₄Ligase enzyme connects product transformed into escherichia coli DH5 α in 16 ℃ of connections, coats on the LB plate that contains acillin (AMP), and 37 ℃ of incubated overnight are observed the dull and stereotyped uniform distribution bacterium colony of going up next day, and this is the transformant that screens; 4. use the SacI enzyme with above-mentioned recombinant expression plasmid linearizing, electricity consumption punching technology imports among the yeast GS118, select positive colony with the MD plate, G418 concentration is the transformed bacteria of 0.5-4.0mg/ml screening multiple copied on the G418-YPD flat board that concentration increases progressively, cultivate the positive colony of selecting with the YPD liquid that contains 0.5%-2% methyl alcohol, induce its secretory protein, continuous induction was gathered in the crops supernatant after 4 days, obtained the recombinant protein of purifying behind 75% ammonium sulfate precipitation, gel filtration chromatography.