Chlamydia pneumoniae recombinant multi-epitope chimeric antigen and application thereof

Abstract

The invention relates to a chlamydia pneumoniae genetic engineering recombinant antigen which is large in expression quantity, good in solubility and natural in conformation, a plurality of immunodominant epitope regions which are not in cross reaction with other chlamydia are adopted for chimeric expression with fusion protein and are mutually spaced through connecting peptides, and when the recombinant antigen is used for immunodetection of a chlamydia pneumoniae antibody through ELISA or chromatography, the recombinant antigen can be used for immunodetection of a chlamydia pneumoniae antibody. The sensitivity and the specificity of the existing kit can be improved.

Description

Technical Field

[0001] The present invention relates to the field of immunoassay, and particularly to a recombinant multi-epitope chimeric antigen of Chlamydia pneumoniae, a gene sequence encoding the recombinant multi-epitope chimeric antigen of Chlamydia pneumoniae, a plasmid, a host cell containing the plasmid, a kit for detecting Chlamydia pneumoniae antibodies prepared from the recombinant multi-epitope chimeric antigen of Chlamydia pneumoniae, and the use of the recombinant multi-epitope chimeric antigen of Chlamydia pneumoniae in detecting human Chlamydia pneumoniae infection. Background Art

[0002] Chlamydia is a Gram-negative pathogen that can pass through a bacterial filter, parasitize inside cells, and has a unique developmental cycle. It is mostly spherical, has a cell wall and a cell membrane. It is a type of G - prokaryotic microorganism that obligately parasitizes for energy inside eukaryotic cells, with a diameter of about 0.3 - 0.5 micrometers. It has no motility and is widely parasitic in humans, mammals, and birds. The genus Chlamydia includes 11 species: Chlamydia trachomatis (hereinafter simply referred to as Ctr, causing human sexually transmitted diseases and eye infections), Chlamydia muridarum (causing diseases in mice and hamsters), Chlamydia suis (infecting pigs), Chlamydia pneumoniae (causing human respiratory tract infections), Chlamydia pecorum (a common pathogen in livestock), Chlamydia psittaci (prevalent in birds and causing human pneumonia), Chlamydia abortus (causing mammalian abortions), Chlamydia felis (a species found in cats), Chlamydia caviae (a species causing infections in guinea pigs), C. avium (including strains from pigeons and parrots), and C. gallinacea (strains from poultry). Those that can infect humans are Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci, and Chlamydia abortus.

[0003] Chlamydia pneumoniae (C.pneumoniae, hereinafter simply referred to as Cpn), also known as Chlamydophila pneumoniae, can cause acute respiratory diseases, including pneumonia, bronchitis, sinusitis, and pharyngitis, etc. Currently, only one serotype has been found. More than 50% of adults worldwide have been infected with Chlamydia pneumoniae, which can cause 10 - 15% of adult and childhood community-acquired pneumonia. Most infected people have no obvious symptoms or mild symptoms, and severe patients will have serious respiratory system diseases. Infections mostly occur from late autumn to winter and during the transition period between spring and summer.

[0004] The IgM antibody of Lobaria appears about 2 to 3 weeks after the onset of the disease, reaches its peak after 4 to 5 weeks, and cannot be detected after 3 to 5 months. The IgG antibody is slowly produced 30 days before the onset of the disease and reaches a stable period about 3 to 4 months after the onset. In cases of reinfection, the IgM antibody may not exist, or even if it does, it may be at a low titer. The IgG antibody appears earlier, within 1 to 2 weeks after the onset. IgM has an earlier serological reaction to Chlamydia pneumoniae, which helps in the rapid diagnosis of acute Chlamydia pneumoniae infection. Currently, the following methods are available for the detection of Chlamydia pneumoniae:

[0005] Pathogen culture method: High technical requirements, easy to inactivate during transportation, low yield, and usually requires repeated blind passage. Antigen detection method: Problems with sample collection, prone to false positives. PCR method: Difficult to achieve rapid detection. Lack of standardization in extraction procedures, primer definition, etc., prone to false negatives. MIF method: The results are affected by the antigen preparations used and the experience of the professionals responsible for reading and interpreting them. Antibody detection method: Problems with cross-reactivity, and the sensitivity and specificity vary greatly depending on the antigen used.

[0006] Since the convenient and rapid detection of Chlamydia pneumoniae requires the batch preparation of high-quality Cpn antigens, and there are several types of antigens prepared by current existing technologies:

[0007] 1. Chlamydia pneumoniae cultures and their lysed extracts: Such as LPS antigen, Chlamydia pneumoniae antigen lacking LPS, COMC antigen, EB antigen. Its disadvantages are complex composition, large batch-to-batch differences, and high false positives.

[0008] 2. Synthetic polypeptide antigens: Such as the peptides (b), (c), (d), and (g) of the 4 variable regions of MOMP mentioned in WO9857981, Israel, 1997, when used in combination, the sensitivity for detecting IgG + IgA antibodies is only 77%. In addition, there are other drawbacks: Most synthetic peptides belong to linear epitopes, generally do not form a higher-level structure, cannot generate conformational epitopes, and their activity is not as good as that of full-length or truncated fragment recombinant expression antigens; the antibodies detected are IgG, and there is no application verification for detecting IgM or total antibodies; the methodology used is ELISA, which can only be operated with an enzyme-labeled instrument in a professional laboratory and cannot achieve rapid detection.

[0009] 3. Recombinant antigens: Most of them use MOMP. Although it accounts for 60% of the outer membrane components of Cpn, due to its 80% homology with Ctr, false positives will occur. In the article Detection of Chlamydia pneumoniae-Specific Antibodies Binding to the VD2 and VD3 Regions of the Major Outer Membrane Protein, 2003, it is mentioned that only the positive sera of C. pneumoniae can recognize the VD2 and VD3 regions of C. pneumoniae MOMP, suggesting the existence of species-specific epitopes. However, the number of specimens used in this article is insufficient to fully prove the absence of cross-reactivity. In the article Preliminary Application of Recombinant Protein in the VD2-VD3 Region of the ompA Gene of Chlamydia pneumoniae in Serological Diagnosis, Zhou Zhou, 2007, it is mentioned that the VD2 and VD3 used by them are in the range of 148-291aa. However, the antigen they used has at least 3 consecutive homologous regions with Ctr, which will cause false positives due to cross-reaction. In the article Chlamydia: what is on the outside does matter, 2020, Arlieke Gitsels, it is mentioned that the amino acid homology of the major outer membrane proteins between different species of Chlamydia reaches 73% - 80%. The research believes that the MOMP protein of Cpn has poor ability to detect antibodies and has serious cross-reaction with Ctr. Most of the proteins they produced are denatured and lose conformational epitopes. Currently, the most research has been done on MOMP, but the feedback results are not good. The main reasons are: one is that the expression system is limited, and prokaryotic expression cannot prepare MOMP antigens with conformational epitopes; the other is that it has cross-reaction with Ctr.

[0010] In summary, the common problems of current Cpn diagnostic antigens are: the preparation method is backward and not conducive to mass production; the selection of antigen segments is single, especially the homologous segments that cross-react with other Chlamydia such as Ctr have not been removed. Using backward synthetic peptides or prokaryotic expression methods, the best conformation of the antigen cannot be displayed, the activity is poor, resulting in low sensitivity and poor specificity. Therefore, it is necessary to develop and prepare genetically engineered recombinant antigens with high expression level, good solubility, natural conformation, excellent sensitivity and specificity for detecting Cpn antibodies by ELISA or chromatography. Summary of the Invention

[0011] Abbreviations of the present invention: Chlamydia pneumoniae can be abbreviated as Cpn, and Chlamydia trachomatis can be abbreviated as Ctr.

[0012] The first object of the present invention is to provide a recombinant multi-epitope chimeric antigen A or B of Chlamydia pneumoniae with high expression level, good solubility and natural conformation: A. having the amino acid sequence shown in SEQ ID NO: 26; B. a protein derived by substituting, deleting or adding one to several amino acid residues in the amino acid sequence of A and without changing its hydrophilicity. It uses multiple immunodominant epitope regions that do not cross-react with other chlamydia for chimeric expression with a fusion protein, and is separated by a linker peptide. When it is used for ELISA or chromatographic immunoassay for detecting Cpn antibodies, it can improve the sensitivity and specificity of existing kits.

[0013] The second object of the present invention is to provide a gene encoding a recombinant multi-epitope chimeric antigen of Chlamydia pneumoniae, which has the nucleotide sequence shown in SEQ ID NO: 27.

[0014] The third object of the present invention is to provide a plasmid containing the gene sequence encoding the recombinant multi-epitope chimeric antigen of Chlamydia pneumoniae.

[0015] The fourth object of the present invention is to provide a host cell transformed / transfected with an expression plasmid of the gene sequence encoding the recombinant multi-epitope chimeric antigen of Chlamydia pneumoniae.

[0016] The fifth object of the present invention is to provide a kit for detecting Chlamydia pneumoniae antibodies prepared from the recombinant multi-epitope chimeric antigen of Chlamydia pneumoniae.

[0017] The sixth object of the present invention is to provide the use of the recombinant multi-epitope chimeric antigen of Chlamydia pneumoniae in detecting human Chlamydia pneumoniae infection.

[0018] At present, there are generally the following contradictions in Chlamydia pneumoniae antigens: on the one hand, since there are thousands of Chlamydia pneumoniae genes, after infecting humans, the antibody spectra produced in different humans and at different infection stages are different, resulting in the inability to detect the antibodies of the vast majority of infected people with a single protein; on the other hand, since the genes of other types of chlamydia such as Cpn and Ctr are highly homologous, some immunogenic proteins are extremely homologous. For example, the homology of their MOMP reaches more than 80%. This has led to the preparation of Cpn recombinant antigens facing double tests of sensitivity and specificity.

[0019] Due to the low sensitivity of a single protein and the high genetic homology within Cpn species, and since only one serotype of Cpn has been discovered so far, there is a possibility of preparing a Cpn recombinant multi-epitope chimeric antigen by chimeric expression of the immunodominant epitope regions of different Cpn proteins. Such Cpn proteins must be able to specifically distinguish Cpn-infected individuals from Ctr-infected individuals to avoid false positives, as described in articles such as Antigenic Characterization of Chlamydia pneumoniae Isolated in Hiroshima, Japan, 1993; Identification of Two Novel Genes Encoding 97-to 99Kilodalton Outer Membrane Proteins of Chlamydia pneumoniae, 1999; Analysis of humoral immune responses to recombinant Chlamydia pneumoniae antigens, 2020, etc. The inventors screened protein sequences with potential diagnostic value from each ORF of the complete Cpn genome published by NCBI. After comprehensive consideration, 12 candidate proteins, namely IncA, MOMP, TARP, YopC, YwbM, pmp2, pmp6, pmp8, pmp11, pmp21, CT618, and 53 kDa, were finally selected for cloning and expression. Eleven full-length proteins were successfully expressed. After comparison, it was found that although each of these 11 full-length proteins had a certain sensitivity when used alone, there was a relatively high false positive rate. The reason for the analysis might be the cross-reaction caused by the homologous regions of their protein sequences with other pathogens such as Ctr.

[0020] Subsequently, the truncated immunodominant epitope region antigens of these 12 target proteins were recombinantly expressed. After comparing the activities again, 6 regions with a synergistic complementary effect on sensitivity were found: IncA(323 - 378), MOMP(209 - 267), TARP(103 - 161), YwbM(68 - 123), pmp6(233 - 291), 53 kDa(290 - 340). Finally, these 6 regions were chimerically expressed into a single Chlamydia pneumoniae recombinant multi-epitope chimeric antigen, CpnChimer6.

[0021] The inventors found that the six antigen fragments in the recombinant multi-epitope chimeric antigen CpnChimer6 of Chlamydia pneumoniae could only detect less than half of the positive serum when used alone before, but after chimeric expression, the vast majority of positive serum can be detected. The recombinant multi-epitope chimeric antigen CpnChimer6 of Chlamydia pneumoniae was used to detect IgG and IgM on an ELISA platform or a chromatography platform, and to detect total antibodies by a double antigen sandwich method, and it had good sensitivity and specificity compared with the control product.

[0022] After retrieving the ORF of the Cpn genome and comparing the differences in the protein sequences of Cpn and Ctr, 12 candidate proteins, IncA, MOMP, TARP, YopC, YwbM, pmp2, pmp6, pmp8, pmp11, pmp21, CT618 and 53kDa, were selected for cloning and expression, and their amino acid residues are shown in the sequences of SEQ ID NO: 1-12.

[0023] The first step is to optimize the codons of the 12 candidate proteins IncA, MOMP, TARP, YopC, YwbM, pmp2, pmp6, pmp8, pmp11, pmp21, CT618 and 53kDa, add appropriate restriction sites at both ends, load the synthesized genes into plasmid vectors, and perform subsequent subcloning operations. Other methods can also be used, such as designing gene primers corresponding to these 12 proteins, and using the genomic DNA of Cpn as a template for PCR amplification to obtain their ORF genes.

[0024] The 12 genes obtained above were digested by enzymes and agarose gel electrophoresis to recover the target gene bands, which were then connected to appropriate expression vectors. Positive clones were identified by PCR, enzyme digestion or sequencing. There are many well-known expression vectors. In the present embodiment, pcDNA3.4-TOPO was used as the starting vector, but other vectors not listed are also feasible.

[0025] The 12 plasmids successfully subcloned above are introduced (transformed / transfected) into expression host cells. The host here can be prokaryotic or eukaryotic, such as E. coli, yeast, human, HEK cells, etc. In order to increase the expression amount, the gene can also be pre-codon species optimized before gene synthesis.

[0026] The 12 plasmids were transformed / transfected into expression host cells, cultured and induced, and then the cells were harvested at an appropriate time. The His tag of the vector was used for affinity chromatography purification, or other methods such as ion exchange were used for purification.

[0027] The 11 purified recombinant proteins (pmp2 was abandoned in this step due to extremely low expression levels) were separately used to coat 96-well plates, and the reactivity with sera from infected individuals and normal individuals was detected by the indirect ELISA method.

[0028] Through comparison and summary, it was found that although each of these 11 full-length proteins had a certain sensitivity when used alone, there were relatively high false positives. The reason might be cross-reactions caused by homologous sequence segments with other pathogens such as Ctr. Therefore, the inventor made the following improvements, selecting the immunodominant epitope segments in each protein that did not have too high homology with other pathogens such as Ctr.

[0029] Computer software was used to predict sequence solubility, hydrophilicity, and surface accessibility to select potential dominant epitope regions; the homology between Cpn and Ctr proteins was analyzed, and regions with too high homology between Cpn and Ctr had to be avoided when selecting target segments. Finally, one immunodominant epitope segment was selected from each of the 12 proteins IncA, MOMP, TARP, YopC, YwbM, pmp2, pmp6, pmp8, pmp11, pmp21, CT618, and 53 kDa, namely IncA(323 - 378), MOMP(209 - 267), TARP(103 - 161), YopC(119 - 175), YwbM(68 - 123), pmp2(309 - 344), pmp6(233 - 291), pmp8(750 - 806), pmp11(645 - 699), pmp21(126 - 183), CT618(199 - 241). These 12 segments were cloned and expressed, and their amino acid residues are shown as in SEQ ID NO:13 - 24.

[0030] The 12 truncated immunodominant epitope segment antigens obtained were separately used to coat 96-well plates, and the reactivity with sera from infected individuals and normal individuals was detected by the indirect ELISA method.

[0031] Through comparison and summary, it was found that the 12 truncated immunodominant epitope segment antigens inherited most of the sensitivity of their respective full-length proteins, but the specificity was greatly improved. Among them, the specificities of the 6 epitope segment antigens of IncA(323 - 378), MOMP(209 - 267), TARP(103 - 161), YwbM(68 - 123), pmp6(233 - 291), and 53 kDa(290 - 340) were above 95%. Therefore, it was considered to perform chimeric expression of these 6 immunodominant epitope segments.

[0032] By means of genetic manipulation, six immunodominant epitope regions, namely IncA(323 - 378), MOMP(209 - 267), TARP(103 - 161), YwbM(68 - 123), pmp6(233 - 291), and 53kDa(290 - 340), were ligated into a plasmid to form a tandem pcDNA-CpnChimer6 clone. Subsequently, similar transformation / transfection and purification operations were carried out to obtain the Chlamydia pneumoniae recombinant multi-epitope chimeric antigen CpnChimer6. Then, the reactivity with sera from infected individuals and normal individuals was detected by the indirect ELISA method.

[0033] The inventors surprisingly found that when the six antigen fragments within this Chlamydia pneumoniae recombinant multi-epitope chimeric antigen CpnChimer6 were used alone before, less than half of the positive sera could be detected, but after chimeric expression, the vast majority of positive sera could be detected. Using this Chlamydia pneumoniae recombinant multi-epitope chimeric antigen CpnChimer6 for detecting IgG on the ELISA platform or chromatography platform, detecting IgM, and detecting total antibodies by the double-antigen sandwich method all showed good sensitivity and specificity compared with the control products.

[0034] Advantages of the present invention: The chimeric expression of the dominant epitope regions of multiple different proteins of Chlamydia pneumoniae has higher sensitivity than individual proteins, and has very low protein sequence homology with other species such as Chlamydia trachomatis, avoiding false positives caused by cross-reactions. The selected regions have strong hydrophilicity, and the use of the excellent fusion protein Sumo can increase the expression level and solubility, and the overall protein has good solubility. The different peptide segments are separated by linker peptides, which is beneficial for purification by Ni column affinity chromatography, beneficial for the exposure of their respective epitopes and full binding with antibodies. Using the HEK expression system has better antigen activity than synthetic peptides or prokaryotic-expressed antigens. It is used for indirect detection of IgG, capture detection of IgM, double-antigen sandwich detection of total antibodies, and can also be used for chromatography, with simple and rapid antibody detection, and good overall sensitivity and specificity, and is uniformly applicable to different detection platforms. Specific embodiments

[0035] The objectives, features, and advantages of the present invention will be further elaborated in detail in conjunction with the embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. For the experimental methods without specific conditions indicated in the following embodiments, they are generally carried out under conventional conditions, such as: Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989); Current Protocols in Molecular Biology (Second Edition) (edited by Lu Shengdong); Guidelines for Protein Purification and Identification, Cold Spring Harbor; Clinical Enzyme Immunoassay Techniques, written by Li Jinming, People's Military Medical Press, 2005; the conditions described in Colloidal Gold Immunochromatographic Diagnostic Test Strip Technology (Third Edition), or the standard methods recommended by the manufacturer.

[0036] Example 1: Preparation of Cpn Candidate Full-Length Proteins

[0037] First, search for the Chlamydia pneumoniae sequences on NCBI and screen the partial ORF sequences of the whole genome of Chlamydophila pneumoniae CWL029 published with the GenBank number: AE001363.1. Finally, 12 proteins, namely IncA, MOMP, TARP, YopC, YwbM, pmp2, pmp6, pmp8, pmp11, pmp21, CT618, and 53 kDa, were selected for cloning and expression, and their amino acid residues are shown in SEQ ID NO: 1-12. The protein sequences SEQ ID NO: 1-12 were entrusted to Shanghai Sangon Biotech Co., Ltd. for gene optimization and synthesis. Each has an EcoRI cleavage site at the N-terminus and a BamHI cleavage site at the C-terminus, and finally, they were loaded onto the puc57 vector and delivered to me. They were named in sequence as: puc57-IncA(Comp), puc57-MOMP(Comp), puc57-TARP(Comp), puc57-YopC(Comp), puc57-YwbM(Comp), puc57-pmp2(Comp), puc57-pmp6(Comp), puc57-pmp8(Comp), puc57-pmp11(Comp), puc57-pmp21(Comp), puc57-CT618(Comp), and puc57-53kDa(Comp).

[0038] To modify the pcDNA3.4 vector with the Sumo fusion protein, a gene named Kozak+IgG SP-Sumo was synthesized, and the sequence is shown in SEQ ID NO:25. It contains a promoter, a signal peptide, and restriction enzyme sites, namely: TCTAGAGGATCGAACCCTTCCCTTAAGCCCCCCGCCGCCACCATGGGGTGGAGCTGCATCATCCTGTTCCTGGTGGCCACCGCCACAGGCGTGCACTCCGGTACCGGCGGCATGTCGGACTCAGAAGTCAATCAAGAAGCTAAGCCAGAGGTCAAGCCAGAAGTCAAGCCTGAGACTCACATCAATTTAAAGGTGTCCGATGGATCTTCAGAAATCTTCTTCAAGATCAAAAAGACCACTCCTTTAAGAAGGCTGATGGAAGCGTTCGCTAAAAGACAGGGTAAGGAAATGGACTCCTTAAGATTCTTGTACGACGGTATTCGCATTCAAGCTGATCAGACCCCTGAAGATTTGGACATGGAGGATAACGATATTATTGAGGCTCACAGAGAACAGATTGGTGGTGGCGGCGAATTCCACCACCACCACCATCACCACCACTGAGGATCCGAGGCCGCCGCCAAGGAAGCCGCCGCCAAGCACCACCACCACCATCACCACCACTGAAAGCTT. The above Kozak+IgG SP-Sumo sequence was synthesized by Shanghai Sangon Biotech Co., Ltd., and the obtained gene was delivered in the plasmid puc57 vector. Subsequently, the plasmid puc57-Kozak+IgG SP-Sumo was double-digested with XbaI and HindIII, and the target gene fragment was recovered by gel electrophoresis and ligated into the plasmid vector pcDNA3.4-TOPO that had been digested with the same XbaI and HindIII. The ligation product was transformed into the DH5α strain, and positive monoclonal colonies identified by PCR were selected for sequencing verification. The successfully modified plasmid vector was named pcDNA-SM.

[0039] Subsequently, each of the 12 genes was separately cloned between the EcoRI and BamHI restriction sites on the plasmid pcDNA-SM. The specific steps are as follows: The plasmid puc57-IncA(Comp) was digested with EcoRI and BamHI, and the target gene fragment was recovered by gel extraction and ligated into the plasmid vector pcDNA-SM that had been digested with the same enzymes. The ligation product was transformed into the DH5α strain, and monoclonal colonies that were positive by PCR identification were selected for sequencing verification to obtain the expression plasmid pcDNA-SM-IncA(Comp). The remaining 11 Cpn genes were successfully cloned into the pcDNA-SM plasmid in the same manner. The DH5α bacteria transformed with the pcDNA-SM-IncA(Comp) plasmid were inoculated into 200 ml of LB medium and cultured overnight at 37°C with a rotation speed of 220 rpm. The cells were collected by centrifugation, and the plasmid was prepared in large quantities using an endotoxin-free plasmid large-scale extraction kit (OMEGA, catalog number: D6926-03) for subsequent transfection. HEK-293 cells were pre-cultured and passaged to 500 ml until the logarithmic growth phase (density approximately 2 - 7×106 cells / ml, survival rate greater than 98%). Then, the Zhuhai Kairui KOP293 transient transfection protein expression system was used for transfection with the transfection reagent / plasmid mixture of PEI / pcDNA-SM-IncA(Comp). The detailed steps are described in its user guide (version 4.0) and will not be elaborated here. After transfection, an expression enhancer was added at 24 h. Starting from 72 h, the cell density and viability were counted daily. When the viability was around 70%, the cells were collected, centrifuged at 5000 rpm, and the supernatant was collected. Affinity chromatography purification was performed using the magnetic beads Ni NTA Magarose Beads from Tianrenhe Biotechnology Co., Ltd. (catalog number: SM02505). Elution was carried out with 20 mM PB pH 7.2, 500 mM NaCl, and 300 mM imidazole. The eluate was dialyzed into 20 mM PBS pH 7.2, and its purity was identified by SDS-PAGE to be approximately 95%. The protein concentration was measured using the BCA kit from Beijing Solarbio (catalog number: PC0020), and finally, 30 mg of the target protein was obtained. The transfection and purification of the remaining 11 plasmids were also carried out in the same way. Although the plasmid construction of the pmp2 full-length gene was successful, its expression level was very low, and insufficient protein could be purified. Therefore, 11 different full-length proteins were obtained.

[0040] The 11 prepared full-length proteins were diluted to 2ug / ml with 20mM CB pH9.6, and 100ul was added to each well to coat the 96-well ELISA plate, and the coating was carried out at 37 degrees for 2h. Take out the ELISA plate, discard the coating solution, wash 5 times with PBST, add 200ul blocking solution to each well, and block at 37 degrees for 2h. Take out the ELISA plate, discard the blocking solution, add the 1:100 diluted serum sample to the plate, 100ul to each well, and react at 37 degrees for 30min. Take out the ELISA plate, discard the sample, wash 5 times with PBST, add 100ul of enzyme-labeled IgG secondary antibody to each well, and react at 37 degrees for 30min. Take out the ELISA plate, discard the enzyme-labeled secondary antibody, wash 5 times with PBST, add 50ul of tetramethylbenzidine and hydrogen peroxide to each well, and react at 37 degrees for 15min. Take out the ELISA plate, add 2M sulfuric acid to each well to terminate the reaction, and put the ELISA plate into the ELISA reader to read the value.

[0041] A total of 69 IgG-positive sera and 117 IgG-negative sera were tested, and these specimens were identified and confirmed by the Chlamydia pneumoniae IgG, IgA and IgM antibody ELISA kit LOY-EIAs (Labsystems OY, Helsinki, Finland) and the Chlamydia pneumoniae MIF kit C.pneumoniae MIF IgG, IgA and IgM (Labsystems OY, Helsinki, Finland). The sensitivity and specificity obtained are shown in Table 1 (Note: IncA (Comp) represents the full-length expressed IncA protein, and the other names are similar):

[0042]

[0043] As can be seen from Table 1, although these 11 full-length proteins have certain sensitivity when used alone, there are relatively high false positives. The reason for this may be the cross-reaction caused by the homology segments of the protein sequences of other pathogens such as Ctr. Therefore, the inventors made the following improvements, selecting immunodominant epitope segments in each protein that are not too homologous to other pathogens such as Ctr, as shown in Example 2.

[0044] Example 2: Preparation of Cpn immunodominant epitope segment antigen

[0045] Predict the solubility of the sequence using the Protein-Sol prediction website https: / / protein-sol.manchester.ac.uk / ; predict the hydrophilicity, surface accessibility, and antigenicity using the Protean component of the DNAStar software package; since the overall genes of Cpn and Ctr species are 81% similar, most antigens cross-react with each other, so analyze the protein homology of Cpn and Ctr using the Megalign component of the DNAStar software package. When selecting the target segment, it is necessary to avoid segments with too high homology. Finally, select an immunodominant epitope segment for each of the 12 proteins, namely IncA, MOMP, TARP, YopC, YwbM, pmp2, pmp6, pmp8, pmp11, pmp21, CT618, and 53 kDa. The sequences are shown in Table 2 below:

[0046] IncA(323-378) LEKMKQEAQKAESEFIACVRDRTFGRRETPPPTTPVVEGDESQEEDEGGTPPVSQP MOMP(209-267) EFQYAQSKPKVEELNVICNVSQFSVNKPKGYKGVAFPLPTDAGVATATGTKSATINYHE TARP(103-161) GRTQRRDSEISSSSDGSSISRTSSNASSGETSRAESSPDLGDLDSLSGSERAEGAEGPE YopC(119-175) ELEERKKPRPERRTTADVKRSPRFLPTQEVEEPVPAASKEQLDSIQVWEEKQNYARR YwbM(68-123) kkspdssqgasgpamqspsgptirptrpappppttgganakrpathgkgrapqppt pmp2(309-344) NSPDTGDNKEYTGTIVFSGEKLTEAEAKDEKNRTSK pmp6(233-291) KEKDSTLDANTGVVTFKSNTAKTGGAWSSDDNLALTGNTQVLFQENKTTGSAAQANNPE pmp8(750-806) YTKNDMDTRYTSYPEAQGSWTNNSGALELGGSLALYLPKEAPFFQGYFPFLKFQAVY pmp11(645-699) KTGDENRKGFRHTSGGYVIGGSAHTPKDDLFTFAFCHLFARDKDCFIAHNNSRTY pmp21(126-183) DLDTRNFSQPTQEPDTSNAVSEKISSDTKENRKDLETEDPSKKSGLKEVSSDLPKSPE CT618(199-241) TRPETISDPENRNKPSAEFAARSKAIRNAFIAWLGDVVDLVCD 53kDa(290-340) LPSDSDRPDLPKSDTLRECEENLGFRPQGYEASYSPEESALRPTVEINGIS

[0047] Entrust the 12 polypeptides in Table 2 above to Shanghai Sangon Biotech Co., Ltd. for gene optimization and synthesis. Each C-terminus is attached with a GGS flexible linker, the N-terminus is attached with an EcoRI restriction site, and the C-terminus is attached with MfeI and BamHI restriction sites. Finally, it is loaded onto the puc57 vector and delivered to me.

[0048] Using the same method as in Example 1, prepare the gene expression plasmids for the immunodominant epitope segments of the above 12 proteins, extract a large amount of plasmids, transform 293 cells, and purify the antigens. Finally, obtain 12 recombinant proteins of immunodominant epitope segments.

[0049] Using the same method as in Example 1, detect 69 IgG-positive sera and 117 IgG-negative sera. The results are shown in Table 3 below:

[0050]

[0051]

[0052] As shown in the above results in Table 3, the antigens of the immunodominant epitope segments inherit most of the sensitivity of their respective full-length proteins, and the specificity is greatly improved. Among them, the specificities of the antigens of the 6 epitope segments, namely IncA(323 - 378), MOMP(209 - 267), TARP(103 - 161), YwbM(68 - 123), pmp6(233 - 291), and 53kDa(290 - 340), are above 95%. Therefore, consider performing chimeric expression on these 6 immunodominant epitope segments as shown in Example 3.

[0053] Example 3 Preparation of Chlamydia pneumoniae Recombinant Multi-Epitope Chimeric Antigen

[0054] The plasmid pcDNA-SM-IncA(323-378) was digested with MfeI and BamHI, and the vector band was recovered by gel extraction. At the same time, the plasmid puc57-MOMP(209-267) was digested with EcoRI and BamHI, and the fragment band was recovered by gel extraction. The two were subjected to a ligation reaction, and the resulting positive clone was called pcDNA-SM-IncA(323-378)-MOMP(209-267). Since MfeI and EcoRI are isocaudamers, the restriction sites at this junction will be blocked, facilitating subsequent fragment ligation. Using the same strategy, the remaining fragments TARP(103-161), YwbM(68-123), pmp6(233-291), 53kDa(290-340) were successively concatenated into the plasmid pcDNA-SM-IncA(323-378)-MOMP(209-267). Finally, a clone incorporating 6 immunodominant epitope regions was obtained, which was abbreviated as the pcDNA-CpnChimer6 clone.

[0055] Using the same method as in Example 1, the plasmid of pcDNA-CpnChimer6 was extracted in large quantities, transfected into 293 cells, and the antigen was purified. Finally, the recombinant multi-epitope chimeric antigen CpnChimer6 of Chlamydia pneumoniae was obtained.

[0056] Example 4 Detection of IgG against the recombinant multi-epitope chimeric antigen of Chlamydia pneumoniae

[0057] 4.1 Detection of IgG by ELISA:

[0058] Using the same method as in Example 1, 69 IgG-positive sera and 117 IgG-negative sera were detected by indirect ELISA. The results are shown in Table 4 below:

[0059]

[0060] 4.2 Detection of IgG by chromatography:

[0061] (1) Preparation of colloidal gold: Add 100 ml of ultrapure water to a conical flask, heat it to boiling on a magnetic heating stirrer, add 1 ml of 1% chloroauric acid (Sigma-Aldrich, catalog number: 16961-25-4) solution, immediately add 1 ml of 1% aqueous sodium citrate (Sigma-Aldrich, catalog number: 6132-04-3) solution after boiling, continue boiling for 10 minutes, and then cool naturally.

[0062] (2) Colloidal gold labeling: Take 10 ml of the above-mentioned colloidal gold and put it into a beaker. While stirring, add 0.1 M K2CO3 to adjust the pH to 7.0, and continue stirring for 5 minutes; add a certain amount of recombinant multi-epitope chimeric antigen CpnChimer6 of Chlamydia pneumoniae and chicken IgY antibody, and continue stirring for 15 - 30 minutes; add 0.1 ml of 10% BSA, and continue stirring for 15 - 30 minutes; centrifuge at 8000 g for 59 minutes, discard the supernatant, resuspend the precipitate with colloidal gold diluent (20 mM PB, 250 mM NaCl, 2% BSA, 1% Sucrose, 0.01% Proclin300), make the volume up to 1 ml, fully mix and store at 4 degrees.

[0063] (3) Preparation of gold-labeled pad: Dilute the above gold-labeled complex 10 times with colloidal gold diluent and soak the glass fiber (from Watman) in it, and bake at 37 degrees for 2 hours to make the gold-labeled pad, which is stored in a dry and sealed manner for later use.

[0064] (4) Coating of nitrocellulose (NC) membrane: Dilute anti-human IgG with detection line diluent (10 mM PBS + 2% sucrose) to 1 mg / ml to make the detection line working solution, and dilute mouse anti-IgY polyclonal antibody with the same diluent to 0.5 mg / ml to make the control line working solution. Use a dotting instrument to draw these two working solutions to the corresponding positions on the nitrocellulose membrane (from Millipore, product number: HF135002), and dry at 37 degrees for 8 hours.

[0065] (5) Assemble the above gold-labeled pad, coated nitrocellulose membrane, absorbent paper, polyester plate, sample pad and other auxiliary materials into a gold-labeled detection kit for Chlamydia pneumoniae antibody.

[0066] (6) Detection method: Add 100 ul of the sample to be tested (for example: serum) to the sample pad, and after standing at room temperature for 5 minutes, judge the result. The judgment criteria are as follows:

[0067] ① Only one band appears in the control region and no band appears in the test region, which is negative (-);

[0068] ② Two bands appear, one of which is located in the control region and the other is located in the test region, indicating positive (+);

[0069] ③ No band appears in the control region, indicating an incorrect operation process or the test card has deteriorated or been damaged. In this case, the instruction manual should be read carefully again and a new test strip should be used to retest.

[0070] The results are shown in Table 5:

[0071]

[0072] It can be seen that the immunochromatographic test strip prepared with the recombinant multi-epitope chimeric antigen of Chlamydia pneumoniae has excellent performance in detecting IgG antibodies.

[0073] Example 5 Detection of IgM with Recombinant Multi-epitope Chimeric Antigen of Chlamydia pneumoniae

[0074] 5.1 Detection of IgM by ELISA:

[0075] Label the recombinant multi-epitope chimeric antigen CpnChimer6 of Chlamydia pneumoniae by the sodium periodate method. Take 5 mg of HRP and dissolve it in 0.5 ml of water, add 0.5 ml of fresh 0.06 M NaIO4 aqueous solution, mix well, and place it at 4 °C for 30 min.

[0076] After taking it out, add 0.5 ml of 0.16 M ethylene glycol aqueous solution, place it at room temperature for 30 min, then add 1 ml of aqueous solution containing 5 mg of purified recombinant multi-epitope chimeric antigen CpnChimer6 of Chlamydia pneumoniae, mix well and put it into a dialysis bag and dialyze against 0.05 M carbonate buffer with pH 9.51 overnight.

[0077] After taking it out, add 0.2 ml of NaBH4 solution (5 mg / ml), and place it in the refrigerator for 2 h.

[0078] Add an equal volume of saturated ammonium sulfate, place it in the refrigerator for 30 min, then centrifuge. Resuspend the precipitate with 2.5 ml of 20 mM PBS, add an equal volume of glycerol, mix well and store.

[0079] Dilute the anti-human IgM antibody with 20 mM PB 7.4 to 0.5 - 4 μg / ml, add 100 μl to each well and coat it on a 96-well enzyme-linked immunosorbent assay (ELISA) plate, and incubate at 37 °C for 2 h.

[0080] Take out the ELISA plate, discard the coating solution, wash it once with PBST, add 200 μl of blocking solution to each well, and incubate at 37 °C for 2 h.

[0081] Take out the ELISA plate, discard the blocking solution, add the diluted Cpn positive specimen to the plate, 100 μl per well, and react at 37 °C for 30 min.

[0082] Take out the ELISA plate, discard the sample, wash it 5 times with PBST, add 100 μl of the recombinant multi-epitope chimeric antigen CpnChimer6 of Chlamydia pneumoniae - HRP diluted 1:10,000 to each well, and react at 37 °C for 30 min.

[0083] Take out the ELISA plate, discard the antigen - HRP, wash it 5 times with PBST, add 50 μl of tetramethylbenzidine and hydrogen peroxide to each well, and react at 37 °C for 15 min.

[0084] Take out the enzyme-linked immunosorbent assay (ELISA) plate, add 2 M sulfuric acid to each well to terminate the reaction, and place the ELISA plate in an ELISA reader to read the values. The results are shown in Table 6 as follows:

[0085]

[0086] 5.2 Chromatography for IgM detection:

[0087] (1) Preparation of colloidal gold: Add 100 ml of ultrapure water to a conical flask, heat it to boiling on a magnetic heating stirrer, add 1 ml of 1% chloroauric acid (Sigma-Aldrich, catalog number: 16961-25-4) solution, immediately add 1 ml of 1% trisodium citrate (Sigma-Aldrich, catalog number: 6132-04-3) aqueous solution after boiling, continue boiling for 10 minutes, and then let it cool naturally.

[0088] (2) Colloidal gold labeling: Take 10 ml of the above-mentioned colloidal gold and put it into a beaker. While stirring, add 0.1 M K2CO3 to adjust the pH to 7.0, and continue stirring for 5 minutes; add a certain amount of recombinant multi-epitope chimeric antigen CpnChimer6 of Chlamydia pneumoniae and chicken IgY antibody, and continue stirring for 15 - 30 minutes; add 0.1 ml of 10% BSA, and continue stirring for 15 - 30 minutes; centrifuge at 8000 g for 59 minutes, discard the supernatant, resuspend the precipitate with colloidal gold diluent (20 mM PB, 250 mM NaCl, 2% BSA, 1% sucrose, 0.01% Proclin300), make the volume up to 1 ml, mix well and store at 4 °C.

[0089] (3) Preparation of gold-labeled pad: Dilute the above gold-labeled complex 10-fold with colloidal gold diluent and soak glass fiber (Watman), dry it at 37 °C for 2 hours to make the gold-labeled pad, and store it in a dry and sealed manner for later use.

[0090] (4) Coating of nitrocellulose (NC) membrane: Dilute anti-human IgM with detection line diluent (10 mM PBS + 2% sucrose) to 1 mg / ml to make the detection line working solution, dilute mouse anti-IgY polyclonal antibody with the same diluent to 0.5 mg / ml to make the control line working solution, and use a dot-blotting instrument to draw these two working solutions to the corresponding positions on the nitrocellulose membrane (Millipore, catalog number: HF135002), and dry at 37 °C for 8 hours.

[0091] (5) Assemble the above gold-labeled pad, coated nitrocellulose membrane, absorbent paper, polyester plate, sample pad and other auxiliary materials into a gold-labeled detection kit for Chlamydia pneumoniae antibody.

[0092] (6) Detection method: Add 100 μl of the sample to be tested (for example: serum) to the sample pad, let it stand at room temperature for 5 minutes, and then judge the result. The judgment criteria are as follows:

[0093] ①Only one band appears in the quality control area and no band appears in the test area, which is negative (-);

[0094] ②Two bands appear, one in the quality control area and the other in the test area, indicating positive (+);

[0095] ③No band appears in the quality control area, indicating an incorrect operation process or the test card has deteriorated or been damaged. In this case, the instruction manual should be read carefully again and the test should be repeated with a new test strip. The results are shown in Table 7:

[0096]

[0097] Example 6 Detection of total antibodies with recombinant multi-epitope chimeric antigen of Chlamydia pneumoniae

[0098] Both the total antibody ELISA method and the chromatography method use the principle of double antigen sandwich method for detection. The recombinant multi-epitope chimeric antigen CpnChimer6 of Chlamydia pneumoniae is used as both the coating antigen and the labeled antigen at the same time.

[0099] 6.1 Detection of total antibodies by double antigen sandwich ELISA method:

[0100] The recombinant multi-epitope chimeric antigen CpnChimer6 of Chlamydia pneumoniae prepared in Example 3 was labeled by the sodium periodate method. The specific steps are shown in Example 5. The recombinant multi-epitope chimeric antigen CpnChimer6 of Chlamydia pneumoniae was diluted to 2 μg / ml with 20 mM CB pH 9.6, and 100 μl was added to each well and coated on a 96-well enzyme-linked immunosorbent assay (ELISA) plate at 37 °C for 2 h. The ELISA plate was taken out, the coating solution was discarded, and it was washed 5 times with PBST. 200 μl of blocking solution was added to each well and blocked at 37 °C for 2 h. The ELISA plate was taken out, the blocking solution was discarded, and the diluted serum sample at 1:100 was added to the plate, 100 μl per well, and reacted at 37 °C for 30 min. The ELISA plate was taken out, the sample was discarded, and it was washed 5 times with PBST. 100 μl of the recombinant multi-epitope chimeric antigen CpnChimer6 antigen-HRP of Chlamydia pneumoniae diluted 1:10,000 was added to each well and reacted at 37 °C for 30 min. The ELISA plate was taken out, the enzyme-labeled secondary antibody was discarded, and it was washed 5 times with PBST. 50 μl of tetramethylbenzidine and hydrogen peroxide were added to each well and reacted at 37 °C for 15 min. The ELISA plate was taken out, 2 M sulfuric acid was added to each well to terminate the reaction, and the ELISA plate was placed in an ELISA reader for reading. The results are shown in Table 8:

[0101]

[0102] 6.2 Detection of total antibodies by double antigen sandwich immunochromatography method:

[0103] (1) Preparation of colloidal gold: Add 100 ml of ultrapure water into a conical flask, heat it to boiling on a magnetic heating stirrer, add 1 ml of 1% chloroauric acid (Sigma-Aldrich, catalog number: 16961-25-4) solution, immediately add 1 ml of 1% trisodium citrate (Sigma-Aldrich, catalog number: 6132-04-3) aqueous solution after boiling, continue boiling for 10 minutes, and then cool it naturally.

[0104] (2) Colloidal gold labeling: Take 10 ml of the above-mentioned colloidal gold and put it into a beaker. While stirring, add 0.1 M K2CO3 to adjust the pH to 7.0, and continue stirring for 5 minutes; add a certain amount of Chlamydia pneumoniae recombinant multi-epitope chimeric antigen CpnChimer6 and chicken IgY antibody, and continue stirring for 15 - 30 minutes; add 0.1 ml of 10% BSA, and continue stirring for 15 - 30 minutes; centrifuge at 8000 g for 59 minutes, discard the supernatant, resuspend the precipitate with colloidal gold diluent (20 mM PB, 250 mM NaCl, 2% BSA, 1% Sucrose, 0.01% Proclin300), make the volume up to 1 ml, mix well and store at 4 °C.

[0105] (3) Preparation of gold-labeled pad: Dilute the above-mentioned gold-labeled complex 10 times with colloidal gold diluent and soak glass fiber (Watman), dry it at 37 °C for 2 hours to make the gold-labeled pad, and store it in a dry and sealed manner for later use.

[0106] (4) Coating of nitrocellulose (NC) membrane: Dilute Chlamydia pneumoniae recombinant multi-epitope chimeric antigen CpnChimer6 to 1 mg / ml with test line diluent (10 mM PBS + 2% sucrose) to make the test line working solution, dilute mouse anti-IgY polyclonal antibody to 0.5 mg / ml with the same diluent to make the control line working solution, use a dot blotting instrument to draw these two working solutions to the corresponding positions on the nitrocellulose membrane (Millipore, catalog number: HF135002), and dry at 37 °C for 8 hours.

[0107] (5) Assemble the above-mentioned gold-labeled pad, coated nitrocellulose membrane, absorbent paper, polyester board, sample pad and other accessories into a gold-labeled detection kit for Chlamydia pneumoniae antibody.

[0108] (6) Detection method: Add 100 ul of the sample to be tested (for example: serum) to the sample pad, and after standing at room temperature for 5 minutes, judge the result. The judgment criteria are as follows.

[0109] ① Only one band appears in the quality control area and no band appears in the test area, which is negative (-);

[0110] ② Two bands appear, one of which is located in the quality control area and the other is located in the test area, indicating positive (+);

[0111] ③ No band appears in the quality control area, indicating an incorrect operation process or that the test card has deteriorated or been damaged. In this case, the instruction manual should be carefully read again and the test should be repeated with a new test strip. The results are shown in Table 9:

[0112]

[0113] To sum up: The recombinant multi-epitope chimeric antigen CpnChimer6 of Chlamydia pneumoniae has the advantages of high sensitivity, good specificity, and good versatility on different detection platforms, and can replace the Cpn antigen in the existing kits.

[0114] There are various embodiments in the present invention, and there are a large number of experimental data in each embodiment, which are not suitable to be listed one by one in the present invention. All kinds of modifications and improvements made by those skilled in the art of this technology field without departing from the principle of the present invention should be within the scope of the claims of the present invention.

Claims

1. A recombinant multi-epitope chimeric antigen of Chlamydia pneumoniae, characterized in that A or B: A. having the amino acid sequence shown in SEQ ID NO: 26; B. a protein derived from the amino acid sequence in A by substitution, deletion or addition of one or several amino acid residues without changing its hydrophilicity.

2. A gene sequence encoding a recombinant multi-epitope chimeric antigen of Chlamydia pneumoniae according to claim 1, characterized in that: The polypeptide has the nucleotide sequence shown in SEQ ID NO:

27.

3. A plasmid, characterized in that: A plasmid containing a gene sequence encoding the recombinant multi-epitope chimeric antigen of Chlamydia pneumoniae according to claim 1.

4. A host cell, characterized in that A host cell containing the plasmid according to claim 4.

5. A Chlamydia pneumoniae antibody detection kit prepared using the Chlamydia pneumoniae recombinant multi-epitope chimeric antigen described in claim 1.

6. Use of the recombinant multi-epitope chimeric antigen of Chlamydia pneumoniae as described in claim 1 in detecting human Chlamydia pneumoniae infection.

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