CN110698545B - Recombinant Rta protein for nasopharyngeal carcinoma detection, kit and application thereof - Google Patents
Recombinant Rta protein for nasopharyngeal carcinoma detection, kit and application thereof Download PDFInfo
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Abstract
The invention relates to an in vitro serological diagnosis technology, in particular to a recombinant Rta protein for detecting nasopharyngeal carcinoma, a kit and application thereof. The amino acid sequence of the recombinant Rta protein for detecting nasopharyngeal carcinoma provided by the present invention is composed of at least two sequences selected from the group consisting of sequence 7, sequence 12, sequence 18, 24, 28, 39, 40, 46, 47, 52 and 56 in tandem, preferably sequence 12, sequence 18, sequence 39, sequence 40 and sequence 56 according to 1: 2: 2: 1: 3 in series. The sensitivity of the recombinant Rta protein in nasopharyngeal carcinoma screening reaches 92%, and the specificity reaches 98%.
Description
Technical Field
The invention relates to the technical field of biotechnology, medical immunology and in-vitro serological diagnosis, in particular to a recombinant Rta protein for detecting nasopharyngeal carcinoma, a kit and application thereof.
Background
Nasopharyngeal carcinoma (NPC) refers to a malignant tumor of the nasopharynx portion occurring between the nasal cavity and the pharynx, accounting for 78.08% of head and neck malignant tumors and 92.99% of upper respiratory tumors (nasopharyngeal carcinoma, laryngeal carcinoma, etc.). The nasopharyngeal carcinoma is easy to cause serious impact on society, economy, labor and families, and has great harmfulness, wherein the nasopharyngeal carcinoma is aged at the strong stage of 35 to 50 years, accounts for 60 percent of the number of people who have the nasopharyngeal carcinoma. Men are more susceptible to nasopharyngeal carcinoma than women, with a ratio of about 3 to 1. Nasopharyngeal carcinoma is second only to cervical and breast cancers among female cancers, and is third. According to the statistics of the world health organization, about 80% of the worldwide nasopharyngeal carcinoma cases occur in China, especially in areas such as Guangdong, Guangxi, Hainan, Fujian, Hunan, Jiangxi, the south of Sichuan, hong Kong, Taiwan and the like, and are listed as three major tumors in China together with esophageal cancer and liver cancer. Meanwhile, there are also a number of patients in southeast Asia, such as Singapore, Malaysia, Philippines, Indonesia, and other countries. Particularly, the incidence of nasopharyngeal carcinoma in Chinese people in various places of the overseas (most of which are from Guangdong, Guangxi and Fujian provinces) is still maintained at a high level, which indicates that the occurrence of nasopharyngeal carcinoma has obvious ethnic sensitivity. The distribution of the nasopharyngeal carcinoma in China has obvious regional difference, and the Zhaoqing, the Buddha mountain and the Guangzhou area in Guangdong and the Guangxi Wuzhou area in Guangxi are mutually connected into a whole, which is the area with the highest incidence of the nasopharyngeal carcinoma in the world. The Guangzhou Zhongshan medical university has generally examined 436,786 people in Zhaoqing Xijiang river basin, and the like, and the prevalence rate of nasopharyngeal carcinoma is up to 56.80/10 ten thousand. Nasopharyngeal carcinoma patients have increased at a rate of about 8 ten thousand patients per year in recent years, and have an increasing trend in northern populations of China. The death rate caused by the nasopharyngeal carcinoma at the middle and late stages is very high, even after treatment, the five-year survival rate is only 20-30 percent on average, while the radiotherapy effect at the early stage of the nasopharyngeal carcinoma is excellent, and the five-year survival rate can reach 90 percent. It can be seen that if nasopharyngeal carcinoma can be diagnosed early, both the therapeutic effect and prognosis can be improved significantly. Therefore, the method for diagnosing and screening the nasopharyngeal carcinoma quickly, accurately and simply has a larger market prospect.
Epstein-Barr virus (EBV) is a common gamma herpes virus, and more than 90% of people worldwide infect the virus, mainly invade B lymphocytes, and are susceptible to epithelial cells (including parotid duct, nasopharynx, cervix and other epithelial cells) and glandular cells. Generally, the infected epstein-barr virus in vivo is latent (recessive infection); however, the latent EB virus genome is activated and EB virus enters a lytic state when stimulated by certain factors. According to the classification criteria for carcinogenic factors by the international center for cancer research (IARC) 1999, EB virus has been well-defined as one of the human carcinogenic factors.
In Hovenia, the expression of main EBV lytic genes in nasopharyngeal biopsy tissues (including tumor tissues and normal tissues) is studied by RT-PCR (reverse transcription-polymerase chain reaction) and found that the expressions of BZLF1, BALF2 and BCLF can be detected in NPC samples and control samples, while the expression of BRLF1 can be detected only in NPC samples but not in normal tissues. This result suggests that EB virus reactivation occurs in both nasopharyngeal carcinoma tissues and normal tissues, but BRLF1 gene expression occurs only in nasopharyngeal carcinoma biopsies, suggesting an important role of BRLF1 gene in the development of nasopharyngeal carcinoma.
Protein products expressed by various genes in the incubation period and the lysis period of the EB virus can cause the reaction of a human immune system to generate corresponding antibodies, and the antibodies are distributed in blood and can be detected by an enzyme-linked immunosorbent assay (ELISA). The EB virus is activated firstly in the splitting phase and is the immediate early gene BRLF1, and the expression product is the Rta protein. Rta is a polymeric protein consisting of 605 amino acids, has a large molecular weight (66594.6 Da), a complex chemical composition, an exposed epitope and strong antigenicity, and has corresponding antibodies in blood. The researchers detected 53 untreated nasopharyngeal carcinoma patients and 53 normal control human serum Rta protein IgG antibodies by using an immunoprecipitation method, and the results showed that 44 of 53 nasopharyngeal carcinoma patients detected IgG-Rta (83%), and only 1 normal human detected IgG-Rta (1.9%), which indicated the great potential of the EB virus Rta protein antibody as a nasopharyngeal carcinoma tumor marker. A plurality of clinical researches at home and abroad show that the detection of the Rta-IgG antibody in blood can be used for the auxiliary diagnosis of nasopharyngeal carcinoma.
The patent CN101153059B describes that EB virus Rta protein is used for nasopharyngeal carcinoma screening, diagnosis and treatment effect prediction, simultaneously, antigenic determinants of the Rta protein are further obtained through software antigenicity analysis, and then fusion protein is expressed separately or in series for nasopharyngeal carcinoma screening, so that the EB virus Rta protein has better sensitivity and specificity. The patent CN104086657B describes that the partial amino acid sequence of the Rta protein of the EB virus and the partial amino acid sequence of the EA protein are expressed in series so as to improve the sensitivity and the specificity for screening nasopharyngeal carcinoma. However, these recombinant proteins have a plurality of epitopes due to their long amino acid sequences, and since the specificities and sensitivities of different epitopes are different, the sensitivity and specificity of the reagents cannot be further improved in the recombinant proteins prepared by these methods.
Disclosure of Invention
Based on the above problems, in order to further improve the sensitivity and specificity of nasopharyngeal carcinoma screening, the invention provides a recombinant protein for nasopharyngeal carcinoma diagnosis, wherein the sensitivity of the protein in the nasopharyngeal carcinoma screening reaches 92%, and the specificity of the protein in the nasopharyngeal carcinoma screening reaches 98%.
The amino acid sequence of the recombinant Rta protein for detecting nasopharyngeal carcinoma provided by the invention is composed of at least two sequences selected from the sequences 7, 12, 18, 24, 28, 39, 40, 46, 47, 52 and 56 in tandem.
Preferably, the amino acid sequence of the recombinant Rta protein is represented by seq id No. 12, seq id No. 18, seq id No. 39, seq id No. 40 and seq id No. 56 according to 1: 2: 2: 1: 3 in series.
Preferably, the amino acid sequence of the recombinant Rta protein is sequence 62 or sequence 63.
The invention also provides a preparation method of any recombinant Rta protein, which comprises the following steps: synthesizing the coding gene of the recombinant Rta protein, constructing an expression vector, transforming escherichia coli, and performing protein expression and purification.
The invention also provides a polypeptide composition for detecting nasopharyngeal carcinoma, which is characterized by consisting of at least two polypeptides with amino acid sequences selected from the group consisting of the polypeptides in the sequence 7, 12, 18, 24, 28, 39, 40, 46, 47, 52 and 56.
Preferably, the polypeptide composition is encoded by a polypeptide having an amino acid sequence according to 1: 2: 2: 1: 3 in a molar ratio.
The invention also provides a preparation method of the polypeptide composition, which is characterized by comprising the steps of synthesizing and uniformly mixing the at least two polypeptides.
The invention also provides a kit comprising any one of the recombinant Rta proteins or the polypeptide composition. Preferably, the kit provides a microplate coated with the recombinant Rta protein or the polypeptide composition. More preferably, the kit further provides a wash solution, a sample diluent (e.g., 1% BSA solution), a negative control serum, a positive control serum, a secondary antibody, a chromogenic substrate, and instructions for kit handling.
Preferably, the working concentration of the recombinant Rta protein or the polypeptide composition is 1.0 μ g/ml. When the recombinant Rta protein or polypeptide composition is used for detecting nasopharyngeal carcinoma by an enzyme-linked immunosorbent assay, the coating concentration is preferably 1.0 mu g/ml, and the secondary antibody is preferably 0.04 mu g/ml goat anti-human IgG.
The application of any recombinant Rta protein or the polypeptide composition or the kit in nasopharyngeal carcinoma detection also belongs to the protection scope of the invention.
According to the invention, the Rta protein is divided into 60 polypeptide sequences, polypeptides with different sequences are utilized to determine a normal physical examination serum sample and a nasopharyngeal carcinoma confirmed patient serum sample, polypeptides (epitope) with high sensitivity and good specificity are screened out, and then the polypeptides (epitope) with different proportions are expressed in series to obtain the recombinant Rta protein. The polypeptide with high sensitivity and good specificity can also be directly mixed into a polypeptide composition for screening nasopharyngeal carcinoma. The recombinant Rta protein and polypeptide composition provided by the invention further improves the accuracy of a detection result under the condition of ensuring the high detection rate of nasopharyngeal carcinoma, achieves the sensitivity of 92% and the specificity of 98%, and breaks through the bottleneck that the detection performance cannot be further improved by the existing Rta protein detection technology.
Detailed Description
The present invention is further illustrated below by reference to examples, which are to be understood as being illustrative and illustrative only and not limiting in any way to the scope of the present invention.
Solutions and reagents
Coating buffer solution: weighing sodium dihydrogen phosphate (NaH)2PO4) (national medicine, 20040799) 0.0624g, disodium hydrogen phosphate (Na)2HPO4) (national medicine, 100203008) 0.716 g. 60ml of ultrapure water is added, after complete dissolution, the pH value is adjusted to 7.4, and the volume is adjusted to 100 ml.
Sealing liquid: 20mM PBS, pH 7.4: weighing sodium dihydrogen phosphate (NaH)2PO4) (national medicine, 20040799) 0.0624g, disodium hydrogen phosphate (Na)2HPO4) (national drug, 100203008) 0.716g, sodium chloride (NaCl) (national drug, 10019308) 0.8g, bovine serum albumin (amresco, S12003C 01) 3 g. 60ml of ultrapure water is added, after complete dissolution, the pH value is adjusted to 7.4, and the volume is adjusted to 100 ml.
Cleaning solution: 20mM PBS, pH 7.4: weighing sodium dihydrogen phosphate (NaH)2PO4) (national medicine, 20040799) 0.0624g, disodium hydrogen phosphate (Na)2HPO4) (national drug, 100203008) 0.716g, sodium chloride (NaCl) (national drug, 10019308) 0.8g, Tveen-20 (sigma, 44112) 50. mu.L. 60ml of ultrapure water is added, after complete dissolution, the pH value is adjusted to 7.4, and the volume is adjusted to 100 ml.
1% BSA solution: 20mM PBS, pH 7.4: weighing sodium dihydrogen phosphate (NaH)2PO4) (national medicine, 20040799) 0.0624g, disodium hydrogen phosphate (Na)2HPO4) (national medicine, 100203008) 0.716g, bovine serum albumin (amresco, S12003C 01) 1 g. 60ml of ultrapure water is added, after complete dissolution, the pH value is adjusted to 7.4, and the volume is adjusted to 100 ml.
Streptavidin: purchased from ancient genetics bioengineering (Nanjing) GmbH, cat #: GSA-10002, 5 mg/ml.
Goat anti-human IgG-HRP (cat # 109-008, antibody concentration 0.8 mg/ml), goat anti-human IgA-HRP (cat # 109-.
Chemiluminescent substrate a and substrate B: purchased from Saimer Feishale science and technology (China) Co., Ltd., cat # 34080.
Rta kit: it is also known as IgG detection kit (enzyme linked immunosorbent assay) for protein antibody of Rta of EB virus produced by Co., Ltd, of Beijing, with product numbers A-0348 and A-0396.
100 normal examination serum samples and 100 confirmed nasopharyngeal carcinoma patient serum samples were provided by the Sterculia tumor Hospital.
The experimental reagents which are not particularly described in the invention are all conventional reagents in the field, and can be prepared according to the conventional method in the field or obtained commercially; the experimental procedures not specifically described are conventional in the art and may be referred to, for example, in the Molecular cloning laboratory Manual (Sambrook J & Russell DW, Molecular cloning: a laboratory Manual, 2001), or the manufacturer's instructions.
Example 1 obtaining of highly sensitive Polypeptides
We split the Rta protein sequence (sequence 61 in the sequence table) into 60 polypeptide sequences (sequences 1-60 in the sequence table), as shown in Table 1.
TABLE 1 60 polypeptide sequences of Rta protein
| Serial number | Sequence of | Serial number | Sequence of |
| SEQ1 | mrpkkdgledflrltpeikkqlgslvsdyc | SEQ31 | ptmplkpgaqsadcgdssssssdsgnsdte |
| SEQ2 | nvlnkeftagsveitlrsykickafineak | SEQ32 | qsereearaeaprlrapksrrtsrpnrgqt |
| SEQ3 | ahgrewgglmatlnicnfwailrnnrvrrr | SEQ33 | pcpsnaeepeqpwiaavhqesderpifphp |
| SEQ4 | aenagndacsiacpivmryvldhlivvtdr | SEQ34 | skptflppvkrkkglrdsregmflpkpeag |
| SEQ5 | ffiqapsnrvmipatigtamykllkhsrvr | SEQ35 | saisdvfegrevcqpkrirpfhppgspwan |
| SEQ6 | aytyskvlgvdraaimasgkqvvehlnrme | SEQ36 | rplpaslaptptgpvhepvgsltpapvprp |
| SEQ7 | kegllsskfkafckwvftypvleemfqtmv | SEQ37 | ldpapavtpeashlledpdeetsqavkalr |
| SEQ8 | ssktghltddvkdvraliktlprasyssha | SEQ38 | emadtvipqkeeaaicgqmdlnhppprghl |
| SEQ9 | gqrsyvsgvlpacllstkskavetpilvsg | SEQ39 | deltttlesmtedlnldspltpelneildt |
| SEQ10 | adrmdeelmgndggashtearysesgqfha | SEQ40 | flndecllhamhistglsifdtslf |
| SEQ11 | ftdeleslpsptmplkpgaqsadcgdssss | SEQ41 | qlgslvsdycnvlnkeftagsveitlrsyk |
| SEQ12 | ssdsgnsdteqsereearaeaprlrapksr | SEQ42 | ickafineakahgrewgglmatlnicnfwa |
| SEQ13 | rtsrpnrgqtpcpsnaeepeqpwiaavhqe | SEQ43 | ilrnnrvrrraenagndacsiacpivmryv |
| SEQ14 | sderpifphpskptflppvkrkkglrdsre | SEQ44 | ldhlivvtdrffiqapsnrvmipatigtam |
| SEQ15 | gmflpkpeagsaisdvfegrevcqpkrirp | SEQ45 | ykllkhsrvraytyskvlgvdraaimasgk |
| SEQ16 | fhppgspwanrplpaslaptptgpvhepvg | SEQ46 | qvvehlnrmekegllsskfkafckwvftyp |
| SEQ17 | sltpapvprpldpapavtpeashlledpde | SEQ47 | vleemfqtmvssktghltddvkdvralikt |
| SEQ18 | etsqavkalremadtvipqkeeaaicgqmd | SEQ48 | lprasysshagqrsyvsgvlpacllstksk |
| SEQ19 | lnhppprghldeltttlesmtedlnldspl | SEQ49 | avetpilvsgadrmdeelmgndggashtea |
| SEQ20 | tpelneildtflndecllhamhistglsifdtslf | SEQ50 | rysesgqfhaftdeleslpsptmplkpgaq |
| SEQ21 | flrltpeikkqlgslvsdycnvlnkeftag | SEQ51 | sadcgdssssssdsgnsdteqsereearae |
| SEQ22 | sveitlrsykickafineakahgrewgglm | SEQ52 | aprlrapksrrtsrpnrgqtpcpsnaeepe |
| SEQ23 | atlnicnfwailrnnrvrrraenagndacs | SEQ53 | qpwiaavhqesderpifphpskptflppvk |
| SEQ24 | iacpivmryvldhlivvtdrffiqapsnrv | SEQ54 | rkkglrdsregmflpkpeagsaisdvfegr |
| SEQ25 | mipatigtamykllkhsrvraytyskvlgv | SEQ55 | evcqpkrirpfhppgspwanrplpaslapt |
| SEQ26 | draaimasgkqvvehlnrmekegllsskfk | SEQ56 | ptgpvhepvgsltpapvprpldpapavtpe |
| SEQ27 | afckwvftypvleemfqtmvssktghltdd | SEQ57 | ashlledpdeetsqavkalremadtvipqk |
| SEQ28 | vkdvraliktlprasysshagqrsyvsgvl | SEQ58 | eeaaicgqmdlnhppprghldeltttlesm |
| SEQ29 | pacllstkskavetpilvsgadrmdeelmg | SEQ59 | tedlnldspltpelneildtflndecllha |
| SEQ30 | ndggashtearysesgqfhaftdeleslps | SEQ60 | mhistglsifdtslf |
60 polypeptide sequences shown in table 1 are artificially synthesized by Compton Biotechnology engineering (Shanghai) Co., Ltd, then 100 normal physical examination serum samples and 100 nasopharyngeal carcinoma confirmed patient serum samples are respectively measured by using the 60 different polypeptides, and Cutoff when 99% of normal physical examination serum samples are judged to be negative and the detection rate (namely sensitivity) of nasopharyngeal carcinoma samples are calculated. And (4) screening a polypeptide sequence with higher sensitivity.
The measurement method is as follows:
(1) coating: coating buffer containing 1.0. mu.g/ml SA (streptavidin, palustridia) 100. mu.l/well was added to a microplate (NUNC, cat 4361) overnight at 2-8 ℃;
(2) and (3) sealing: throwing off the coating buffer solution, adding 200 mul/hole sealing solution, and standing overnight at 2-8 ℃;
(3) and (3) drying: throwing off the confining liquid, and drying at 30 ℃ for 4 hours;
(4) sample adding: add 1.0. mu.g/ml biotin-labeled polypeptide solution (SEQ 1-SEQ60, biologies) (1% BSA solution dilution), 100. mu.L/well;
(5) sample adding: adding a sample to be detected, and 10 mu L/hole;
(6) and (3) incubation: 30min at 37 ℃;
(7) washing: throwing off the liquid in the microporous plate, adding cleaning solution, and cleaning for 5 times at a rate of 300 mu L per hole;
(8) sample adding: goat anti-human IgG-HRP (purchased from Jackson) (diluted with 1% BSA solution at 1/20000 dilution ratio) was added at 100. mu.L/well;
(9) and (3) incubation: 15min at 37 ℃;
(10) washing: throwing off the liquid in the microporous plate, adding cleaning solution, and cleaning for 5 times at a rate of 300 mu L per hole;
(11) substrate: adding chemiluminescence substrate A and substrate B (Thermo, cat No. 34080), reacting at 50 μ L/well in dark for 5min, and measuring luminescence value (Xiamen Sendong ECLIA-IIS type chemiluminescence immunoassay analyzer) with luminescence intensity expressed in Relative Light Unit (RLU).
And (4) judging a result: RLU when at least 99% of the normal examination serum samples are judged to be negative is defined as Cutoff value, and then the detection rate of nasopharyngeal carcinoma samples, namely sensitivity, is calculated. The results show that the sensitivity of SEQ12 (77%), SEQ18 (72%), SEQ39 (82%), SEQ40 (73%), SEQ56 (76%) was higher (table 2).
TABLE 2.60 specificity and sensitivity of the polypeptides for nasopharyngeal carcinoma
| Antigen sequences | Specificity of | Sensitivity of the composition | Cutoff | Antigen sequences | Specificity of | Sensitivity of the composition | Cutoff |
| Rta kit | 99% | 84% | 21000 | SEQ31 | 99% | 51% | 71600 |
| SEQ1 | 99% | 54% | 59960 | SEQ32 | 99% | 64% | 40350 |
| SEQ2 | 99% | 45% | 40670 | SEQ33 | 99% | 53% | 60550 |
| SEQ3 | 99% | 67% | 65140 | SEQ34 | 99% | 40% | 63850 |
| SEQ4 | 99% | 55% | 45420 | SEQ35 | 99% | 57% | 16220 |
| SEQ5 | 99% | 65% | 41880 | SEQ36 | 99% | 37% | 39080 |
| SEQ6 | 99% | 46% | 26680 | SEQ37 | 99% | 39% | 72800 |
| SEQ7 | 99% | 70% | 52280 | SEQ38 | 99% | 44% | 34240 |
| SEQ8 | 99% | 57% | 52820 | SEQ39 | 99% | 82% | 35060 |
| SEQ9 | 99% | 45% | 20960 | SEQ40 | 99% | 73% | 30960 |
| SEQ10 | 99% | 55% | 62200 | SEQ41 | 99% | 60% | 34400 |
| SEQ11 | 99% | 53% | 44590 | SEQ42 | 99% | 40% | 56680 |
| SEQ12 | 99% | 77% | 23370 | SEQ43 | 99% | 43% | 40140 |
| SEQ13 | 99% | 48% | 39890 | SEQ44 | 99% | 60% | 30500 |
| SEQ14 | 99% | 37% | 35870 | SEQ45 | 99% | 57% | 58030 |
| SEQ15 | 99% | 48% | 61920 | SEQ46 | 99% | 71% | 74810 |
| SEQ16 | 99% | 67% | 34770 | SEQ47 | 99% | 70% | 33390 |
| SEQ17 | 99% | 50% | 23520 | SEQ48 | 99% | 55% | 31140 |
| SEQ18 | 99% | 72% | 37430 | SEQ49 | 99% | 67% | 19980 |
| SEQ19 | 99% | 57% | 33300 | SEQ50 | 99% | 42% | 73920 |
| SEQ20 | 99% | 64% | 32460 | SEQ51 | 99% | 55% | 38450 |
| SEQ21 | 99% | 62% | 70490 | SEQ52 | 99% | 72% | 32980 |
| SEQ22 | 99% | 36% | 69220 | SEQ53 | 99% | 67% | 43610 |
| SEQ23 | 99% | 60% | 48590 | SEQ54 | 99% | 45% | 61550 |
| SEQ24 | 99% | 70% | 60440 | SEQ55 | 99% | 40% | 71250 |
| SEQ25 | 99% | 50% | 64130 | SEQ56 | 99% | 76% | 58480 |
| SEQ26 | 99% | 59% | 33630 | SEQ57 | 99% | 38% | 73170 |
| SEQ27 | 99% | 56% | 34640 | SEQ58 | 99% | 50% | 47200 |
| SEQ28 | 99% | 71% | 49060 | SEQ59 | 99% | 59% | 61590 |
| SEQ29 | 99% | 62% | 65550 | SEQ60 | 99% | 42% | 21390 |
| SEQ30 | 99% | 54% | 18800 |
Note: the Rta kit is an EB virus Rta protein antibody IgG detection kit (enzyme-linked immunosorbent assay) produced by the same Xin biotechnology (Beijing) Limited company, the product number is A-0348/A-0396, and the national mechanical Standard is 20173400657. Specific calculation formula: specificity% = number of normal physical examination serum samples judged to be negative/number of normal physical examination serum samples.
Example 2 combination of polypeptide sequences
By comparing the measurement results in example 1, the polypeptide sequences with higher sensitivity are selected, the selected sequences are mixed according to the ratio of Cutoff value RLU to prepare mixed polypeptide sequences, and then the sensitivity and specificity of the reagent are further evaluated by measuring 100 normal physical examination serum samples and 100 nasopharyngeal carcinoma confirmed patient serum samples, and the measuring method is the same as that in example 1.
The results show that the selected polypeptide sequences SEQ12, SEQ18, SEQ39, SEQ40, SEQ56 are according to SEQ 12: SEQ 18: SEQ 39: SEQ 40: SEQ56 ═ 1: 2: 2: 1: 3, the mixture has very good clinical diagnosis performance, and the specificity and the sensitivity are respectively 98% and 92% (table 3).
TABLE 3 determination of specificity and sensitivity of Mixed Polypeptides
| Numbering | SEQ12：SEQ18：SEQ39：SEQ40：SEQ56 | Specificity of | Sensitivity of the composition |
| Ratio 1 | 1：1：1：1：1 | 97% | 79% |
| Ratio 2 | 1：1：1：1：2 | 97% | 81% |
| Ratio 3 | 1：2：2：1：2 | 97% | 89% |
| Ratio 4 | 1：2：2：1：3 | 98% | 92% |
Example 3 tandem expression of polypeptide sequences
Escherichia coli (E.coli) available from Beijing Yiqian Shenzhou Biotechnology Co., LtdE.coli) Expression system for the following proteins Pro1 (SEQ ID NO: 62), Pro2 (SEQ ID NO: 63),Pro3 (SEQ ID NO: 64) for gene synthesis, expression vector construction, protein expression and purification. Wherein, a target gene segment is obtained by carrying out gene synthesis on DNA sequences of proteins to be expressed, namely Pro1, Pro2 and Pro 3; connecting the target gene fragment with the plasmid by adopting ligase to obtain an expression vector; transforming escherichia coli by adopting a heat shock method to obtain recombinant bacteria; culturing the recombinant bacteria, and performing protein expression; the expressed protein is subjected to affinity purification by adopting a nickel column and a glutathione column to obtain target proteins Pro1, Pro2 and Pro 3.
Pro1：SEQ12-SEQ18-SEQ18-SEQ39-SEQ39-SEQ40-SEQ56-SEQ56-SEQ56-His-GST；
Pro2：SEQ12-SEQ18-SEQ39-SEQ40-SEQ56-SEQ18-SEQ39-SEQ56-SEQ56-His-GST；
Pro3：SEQ12-SEQ18-SEQ39-SEQ40-SEQ56-His-GST。
Pro1, Pro2 and Pro3 proteins are respectively used for coating a microporous plate, and each 100 cases of a normal physical examination serum sample and a nasopharyngeal carcinoma patient serum sample are measured, wherein the method comprises the following steps:
(1) coating: coating buffer containing 1.0 μ g/ml of target protein (Pro 1/Pro 2/Pro 3), 100 μ l/well, adding into a microplate (NUNC, cat 4361), and standing at 2-8 deg.C overnight;
(2) and (3) sealing: throwing off the coating buffer solution, adding the confining liquid, 200 mu l/hole, and standing overnight at 2-8 ℃;
(3) and (3) drying: throwing off the confining liquid, and drying at 30 ℃ for 4 hours;
(4) adding a sample diluent: adding 1% BSA solution, 100. mu.L/well;
(5) sample adding: adding a sample to be detected, and 10 mu L/hole;
(6) and (3) incubation: 30min at 37 ℃;
(7) washing: throwing off the liquid in the microporous plate, adding cleaning solution, and cleaning for 5 times at a rate of 300 mu L per hole;
(8) sample adding: goat anti-human IgG-HRP (purchased from Jackson, cat # 109-;
(9) and (3) incubation: 15min at 37 ℃;
(10) washing: throwing off the liquid in the microporous plate, adding cleaning solution, and cleaning for 5 times at a rate of 300 mu L per hole;
(11) substrate: adding chemiluminescence substrate A and substrate B (Thermo, cat No. 34080), reacting at 50 μ L/well in dark for 5min, and measuring luminescence value (Xiamen Sendong ECLIA-IIS type chemiluminescence immunoassay analyzer) with luminescence intensity expressed in Relative Light Unit (RLU).
And (4) judging a result: when at least 99% of the normal examination serum samples are judged to be negative, the detection rate of the nasopharyngeal carcinoma samples is the sensitivity. The results show that the sensitivity and specificity of the protein Pro1 is comparable to that of Pro2, and the sensitivity of the protein Pro3 is poor (Table 4).
TABLE 4 results of specificity and sensitivity assays for three recombinant proteins
| Recombinant proteins | Specificity of | Sensitivity of the composition |
| Pro1 | 98% | 92% |
| Pro2 | 98% | 91% |
| Pro3 | 97% | 78% |
Example 4 preparation of a kit for nasopharyngeal carcinoma detection
1. Selection of coating protein and Secondary antibody
The microplate was coated with each of the proteins Pro1, Pro2 and Pro3 prepared in example 3, and 100 normal physical examination serum samples and 100 nasopharyngeal carcinoma patient serum samples were examined to measure specific IgG, IgA, IgM, IgG + IgM, and IgG + IgM + IgA, respectively, and to screen for a coating protein and a secondary antibody having high sensitivity.
The measurement method is as follows:
(1) coating: coating buffer containing 1.0 μ g/ml of target protein (Pro 1/Pro 2/Pro 3), 100 μ l/well, adding into a microplate (NUNC, cat 4361), and standing at 2-8 deg.C overnight;
(2) and (3) sealing: throwing off the coating buffer solution, adding the confining liquid, 200 mu l/hole, and standing overnight at 2-8 ℃;
(3) and (3) drying: throwing off the confining liquid, and drying at 30 ℃ for 4 hours;
(4) adding a sample diluent: adding 1% BSA solution, 100. mu.L/well;
(5) sample adding: adding a sample to be detected, and 10 mu L/hole;
(6) and (3) incubation: 30min at 37 ℃;
(7) washing: throwing off the liquid in the microporous plate, adding cleaning solution, and cleaning for 5 times at a rate of 300 mu L per hole;
(8) sample adding: adding 100 mu L/hole of goat anti-human IgG-HRP/goat anti-human IgA-HRP/goat anti-human IgM-HRP/goat anti-human (IgG + IgM) -HRP/goat anti-human (IgG + IgM + IgA) -HRP (diluted by 1% BSA solution with the dilution ratio of 1/20000);
(9) and (3) incubation: 15min at 37 ℃;
(10) washing: throwing off the liquid in the microporous plate, adding cleaning solution, and cleaning for 5 times at a rate of 300 mu L per hole;
(11) substrate: adding chemiluminescence substrate A and substrate B (Thermo, cat No. 34080), reacting at 50 μ L/well in dark for 5min, and measuring luminescence value (Xiamen Sendong ECLIA-IIS type chemiluminescence immunoassay analyzer) with luminescence intensity expressed in Relative Light Unit (RLU).
And (4) judging a result: when at least 98% of normal physical examination serum samples are judged to be negative, the detection rate of nasopharyngeal carcinoma samples, namely sensitivity, is higher, and the kit performance is better. The results show that: the kit has better sensitivity when Pro1 or Pro2 is used as the coating protein to measure specific IgG (Table 5).
TABLE 5 specificity and sensitivity results of the kit
| Recombinant proteins | Second antibody | Specificity of | Sensitivity of the composition |
| Pro1 | Sheep anti-human IgG-HRP | 98% | 92% |
| Pro1 | Sheep anti-human IgM-HRP | 98% | 20% |
| Pro1 | Sheep anti-human IgA-HRP | 98% | 45% |
| Pro1 | Sheep anti-human (IgG + IgM) -HRP | 98% | 76% |
| Pro1 | Sheep anti-human (IgG + IgM + IgA) -HRP | 98% | 85% |
| Pro2 | Sheep anti-human IgG-HRP | 98% | 91% |
| Pro2 | Sheep anti-human IgM-HRP | 98% | 23% |
| Pro2 | Sheep anti-human IgA-HRP | 98% | 42% |
| Pro2 | Sheep anti-human (IgG + IgM) -HRP | 98% | 78% |
| Pro2 | Sheep anti-human (IgG + IgM + IgA) -HRP | 98% | 84% |
| Pro3 | Sheep anti-human IgG-HRP | 98% | 75% |
| Pro3 | Sheep anti-human IgM-HRP | 98% | 19% |
| Pro3 | Sheep anti-human IgA-HRP | 98% | 35% |
| Pro3 | Sheep anti-human (IgG + IgM) -HRP | 98% | 53% |
| Pro3 | Sheep anti-human (IgG + IgM + IgA) -HRP | 98% | 68% |
2. Selection of protein coating concentration and concentration of secondary antibody
The protein Pro1, Pro2 and Pro3 are respectively used for coating the microporous plate, 100 normal physical examination serum samples and 100 nasopharyngeal carcinoma patient serum samples are measured, and different coating protein concentrations and goat anti-human IgG-HRP concentrations are set.
The measurement method is as follows:
(1) coating: coating buffer containing 0.01-10 μ g/ml of target protein (Pro 1/Pro 2/Pro 3), 100 μ l/well, adding into microwell plate (NUNC, cat 4361), and standing at 2-8 deg.C overnight;
(2) and (3) sealing: throwing off the coating buffer solution, adding the confining liquid, 200 mu l/hole, and standing overnight at 2-8 ℃;
(3) and (3) drying: throwing off the confining liquid, and drying at 30 ℃ for 4 hours;
(4) adding a sample diluent: adding 1% BSA solution, 100. mu.L/well;
(5) sample adding: adding a sample to be detected, and 10 mu L/hole;
(6) and (3) incubation: 30min at 37 ℃;
(7) washing: throwing off the liquid in the microporous plate, adding cleaning solution, and cleaning for 5 times at a rate of 300 mu L per hole;
(8) sample adding: adding 100 mu L/well of goat anti-human IgG-HRP (Jackson product, cat # 109-;
(9) and (3) incubation: 15min at 37 ℃;
(10) washing: throwing off the liquid in the microporous plate, adding cleaning solution, and cleaning for 5 times at a rate of 300 mu L per hole;
(11) substrate: adding chemiluminescence substrate A and substrate B (Thermo, cat No. 34080), reacting at 50 μ L/well in dark for 5min, and measuring luminescence value (Xiamen Sendong ECLIA-IIS type chemiluminescence immunoassay analyzer) with luminescence intensity expressed in Relative Light Unit (RLU).
And (4) judging a result: when at least 98% of normal physical examination serum samples are judged to be negative, the detection rate of nasopharyngeal carcinoma samples, namely sensitivity, is higher, and the kit performance is better. The results show that the kit performance is optimal when the protein coating concentration is 1.0 mug/ml and the working concentration of the goat anti-human IgG-HRP is 0.04 mug/ml (Table 6).
TABLE 6 results of specificity and sensitivity measurements for different coating protein concentrations
| Recombinant proteins | Concentration of coating protein | Sheep anti-human IgG-HRP dilution ratio | Specificity of | Sensitivity of the composition |
| Pro1 | 0.01 μg/ml | 1/1k | 98% | 75% |
| Pro1 | 0.1 μg/ml | 1/5k | 98% | 85% |
| Pro1 | 1.0 μg/ml | 1/2w | 98% | 92% |
| Pro1 | 10 μg/ml | 1/5w | 98% | 91% |
| Pro2 | 0.01 μg/ml | 1/1k | 98% | 76% |
| Pro2 | 0.1 μg/ml | 1/5k | 98% | 86% |
| Pro2 | 1.0 μg/ml | 1/2w | 98% | 91% |
| Pro2 | 10 μg/ml | 1/5w | 98% | 89% |
| Pro3 | 0.01 μg/ml | 1/1k | 98% | 61% |
| Pro3 | 0.1 μg/ml | 1/5k | 98% | 70% |
| Pro3 | 1.0 μg/ml | 1/2w | 98% | 75% |
| Pro3 | 10 μg/ml | 1/5w | 98% | 74% |
Note: 1/1k, 1/5k, 1/2w and 1/5w respectively represent the dilution of goat anti-human IgG-HRP antibody (Jackson product, cat # 109. sub.035. sub.008. sub.antibody concentration 0.8 mg/ml) at the ratio of 1:1000, 1:5000, 1:20000 and 1: 50000.
3. Assembly of the kit
Kit 1: the Pro1 protein-coated microplate (the preparation method is the same as the steps (1) to (3) of the determination method in the selection of the protein coating concentration and the concentration of the secondary antibody), a reagent bottle containing a cleaning solution, a reagent bottle containing 1% BSA, a serum tube containing a negative control, a serum tube containing a positive control, a reagent bottle containing goat anti-human IgG-HRP, a reagent bottle containing a chemiluminescent substrate A, a reagent bottle containing a chemiluminescent substrate B and a kit use instruction are all put into the kit.
And (3) kit 2: the Pro2 protein-coated microplate (the preparation method is the same as the steps (1) to (3) of the determination method in the selection of the protein coating concentration and the concentration of the secondary antibody), a reagent bottle containing a cleaning solution, a reagent bottle containing 1% BSA, a serum tube containing a negative control, a serum tube containing a positive control, a reagent bottle containing goat anti-human IgG-HRP, a reagent bottle containing a chemiluminescent substrate A, a reagent bottle containing a chemiluminescent substrate B and a kit use instruction are all put into the kit.
Kit 3: the Pro3 protein-coated microplate (the preparation method is the same as the steps (1) to (3) of the determination method in the selection of the protein coating concentration and the concentration of the secondary antibody), a reagent bottle containing a cleaning solution, a reagent bottle containing 1% BSA, a serum tube containing a negative control, a serum tube containing a positive control, a reagent bottle containing goat anti-human IgG-HRP, a reagent bottle containing a chemiluminescent substrate A, a reagent bottle containing a chemiluminescent substrate B and a kit use instruction are all put into the kit.
In the above kit, goat anti-human IgG-ALP (alkaline phosphatase) was used in place of goat anti-human IgG-HRP, and BCIP/NBT was used in place of chemiluminescent substrate A and substrate B, respectively.
Kit operating instructions
1) To the plate was added 1% BSA solution at 100. mu.L/well.
2) Adding the sample to be tested/negative control/positive control, 10 mu L/hole; incubate at 37 ℃ for 30 min.
3) And (3) throwing off the liquid in the microporous plate, adding a cleaning solution, and cleaning for 5 times at a concentration of 300 mu L per hole.
4) Adding 100 μ L/well of goat anti-human IgG-HRP (diluted with 1% BSA solution at 1/20000); incubate at 37 ℃ for 15 min.
5) And (3) throwing off the liquid in the microporous plate, adding a cleaning solution, and cleaning for 5 times at a concentration of 300 mu L per hole.
6) Adding chemiluminescence substrate A and substrate B at 50 μ L/well, and reacting for 5min in dark.
7) Luminescence values were measured and luminescence intensities are expressed in Relative Light Units (RLU).
8) And (4) judging a result: and if the measurement value of the sample to be measured is more than or equal to the positive control measurement value/3, the sample is judged to be positive, otherwise, the sample is judged to be negative.
Sequence listing
<110> Co Xin Biotechnology (Beijing) Ltd
<120> recombinant Rta protein for nasopharyngeal carcinoma detection, kit and application thereof
<130>P190857-TXS
<160>64
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Met Arg Pro Lys Lys Asp Gly Leu Glu Asp Phe Leu Arg Leu Thr Pro
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Glu Ile Lys Lys Gln Leu Gly Ser Leu Val Ser Asp Tyr Cys
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Asn Val Leu Asn Lys Glu Phe Thr Ala Gly Ser Val Glu Ile Thr Leu
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Arg Ser Tyr Lys Ile Cys Lys Ala Phe Ile Asn Glu Ala Lys
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Ala His Gly Arg Glu Trp Gly Gly Leu Met Ala Thr Leu Asn Ile Cys
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Asn Phe Trp Ala Ile Leu Arg Asn Asn Arg Val Arg Arg Arg
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Ala Glu Asn Ala Gly Asn Asp Ala Cys Ser Ile Ala Cys Pro Ile Val
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Met Arg Tyr Val Leu Asp His Leu Ile Val Val Thr Asp Arg
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Phe Phe Ile Gln Ala Pro Ser Asn Arg Val Met Ile Pro Ala Thr Ile
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Leu Ile Lys Thr Leu Pro Arg Ala Ser Tyr Ser Ser His Ala
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Gly Gln Arg Ser Tyr Val Ser Gly Val Leu Pro Ala Cys Leu Leu Ser
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Ala Asp Arg Met Asp Glu Glu Leu Met Gly Asn Asp Gly Gly Ala Ser
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His Thr Glu Ala Arg Tyr Ser Glu Ser Gly Gln Phe His Ala
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Phe Thr Asp Glu Leu Glu Ser Leu Pro Ser Pro Thr Met Pro Leu Lys
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Pro Gly Ala Gln Ser Ala Asp Cys Gly Asp Ser Ser Ser Ser
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Ser Ser Asp Ser Gly Asn Ser Asp Thr Glu Gln Ser Glu Arg Glu Glu
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Ala Arg Ala Glu Ala Pro Arg Leu Arg Ala Pro Lys Ser Arg
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Arg Thr Ser Arg Pro Asn Arg Gly Gln Thr Pro Cys Pro Ser Asn Ala
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Glu Glu Pro Glu Gln Pro Trp Ile Ala Ala Val His Gln Glu
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Ser Asp Glu Arg Pro Ile Phe Pro His Pro Ser Lys Pro Thr Phe Leu
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Pro Pro Val Lys Arg Lys Lys Gly Leu Arg Asp Ser Arg Glu
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Gly Met Phe Leu Pro Lys Pro Glu Ala Gly Ser Ala Ile Ser Asp Val
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Phe Glu Gly Arg Glu Val Cys Gln Pro Lys Arg Ile Arg Pro
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Phe His Pro Pro Gly Ser Pro Trp Ala Asn Arg Pro Leu Pro Ala Ser
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Leu Ala Pro Thr Pro Thr Gly Pro Val His Glu Pro Val Gly
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Ser Leu Thr Pro Ala Pro Val Pro Arg Pro Leu Asp Pro Ala Pro Ala
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Val Thr Pro Glu Ala Ser His Leu Leu Glu Asp Pro Asp Glu
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Glu Thr Ser Gln Ala Val Lys Ala Leu Arg Glu Met Ala Asp Thr Val
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Ile Pro Gln Lys Glu Ala Ala Ile Cys Gly Gln Met Asp
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Leu Asn His Pro Pro Pro Arg Gly His Leu Asp Glu Leu Thr Thr Thr
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Leu Glu Ser Met Thr Glu Asp Leu Asn Leu Asp Ser Pro Leu
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Thr Pro Glu Leu Asn Glu Ile Leu Asp Thr Phe Leu Asn Asp Glu Cys
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Leu Leu His Ala Met His Ile Ser Thr Gly Leu Ser Ile Phe Asp Thr
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Ser Leu Phe
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Phe Leu Arg Leu Thr Pro Glu Ile Lys Lys Gln Leu Gly Ser Leu Val
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Ser Asp Tyr Cys Asn Val Leu Asn Lys Glu Phe Thr Ala Gly
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Ser Val Glu Ile Thr Leu Arg Ser Tyr Lys Ile Cys Lys Ala Phe Ile
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Asn Glu Ala Lys Ala His Gly Arg Glu Trp Gly Gly Leu Met
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Ala Thr Leu Asn Ile Cys Asn Phe Trp Ala Ile Leu Arg Asn Asn Arg
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Val Arg Arg Arg Ala Glu Asn Ala Gly Asn Asp Ala Cys Ser
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Ile Ala Cys Pro Ile Val Met Arg Tyr Val Leu Asp His Leu Ile Val
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Val Thr Asp Arg Phe Phe Ile Gln Ala Pro Ser Asn Arg Val
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Met Ile Pro Ala Thr Ile Gly Thr Ala Met Tyr Lys Leu Leu Lys His
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Ser Arg Val Arg Ala Tyr Thr Tyr Ser Lys Val Leu Gly Val
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Asp Arg Ala Ala Ile Met Ala Ser Gly Lys Gln Val Val Glu His Leu
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Asn Arg Met Glu Lys Glu Gly Leu Leu Ser Ser Lys Phe Lys
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Val Lys Asp Val Arg Ala Leu Ile Lys Thr Leu Pro Arg Ala Ser Tyr
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Ser Ser His Ala Gly Gln Arg Ser Tyr Val Ser Gly Val Leu
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Val Lys Asp Val Arg Ala Leu Ile Lys Thr Leu Pro Arg Ala Ser Tyr
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Ser Ser His Ala Gly Gln Arg Ser Tyr Val Ser Gly Val Leu
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Pro Ala Cys Leu Leu Ser Thr Lys Ser Lys Ala Val Glu Thr Pro Ile
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Leu Val Ser Gly Ala Asp Arg Met Asp Glu Glu Leu Met Gly
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Asn Asp Gly Gly Ala Ser His Thr Glu Ala Arg Tyr Ser Glu Ser Gly
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Gln Phe His Ala Phe Thr Asp Glu Leu Glu Ser Leu Pro Ser
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Pro Thr Met Pro Leu Lys Pro Gly Ala Gln Ser Ala Asp Cys Gly Asp
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Ser Ser Ser Ser Ser Ser Asp Ser Gly Asn Ser Asp Thr Glu
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Gln Ser Glu Arg Glu Glu Ala Arg Ala Glu Ala Pro Arg Leu Arg Ala
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Pro Lys Ser Arg Arg Thr Ser Arg Pro Asn Arg Gly Gln Thr
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Pro Cys Pro Ser Asn Ala Glu Glu Pro Glu Gln Pro Trp Ile Ala Ala
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Val His Gln Glu Ser Asp Glu Arg Pro Ile Phe Pro His Pro
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Ser Lys Pro Thr Phe Leu Pro Pro Val Lys Arg Lys Lys Gly Leu Arg
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Asp Ser Arg Glu Gly Met Phe Leu Pro Lys Pro Glu Ala Gly
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Ser Ala Ile Ser Asp Val Phe Glu Gly Arg Glu Val Cys Gln Pro Lys
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Arg Ile Arg Pro Phe His Pro Pro Gly Ser Pro Trp Ala Asn
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Arg Pro Leu Pro Ala Ser Leu Ala Pro Thr Pro Thr Gly Pro Val His
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Glu Pro Val Gly Ser Leu Thr Pro Ala Pro Val Pro Arg Pro
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Leu Asp Pro Ala Pro Ala Val Thr Pro Glu Ala Ser His Leu Leu Glu
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Asp Pro Asp Glu Glu Thr Ser Gln Ala Val Lys Ala Leu Arg
20 25 30
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Glu Met Ala Asp Thr Val Ile Pro Gln Lys Glu Glu Ala Ala Ile Cys
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Gly Gln Met Asp Leu Asn His Pro Pro Pro Arg Gly His Leu
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Asp Glu Leu Thr Thr Thr Leu Glu Ser Met Thr Glu Asp Leu Asn Leu
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Asp Ser Pro Leu Thr Pro Glu Leu Asn Glu Ile Leu Asp Thr
20 25 30
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<213> Artificial Sequence (Artificial Sequence)
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Phe Leu Asn Asp Glu Cys Leu Leu His Ala Met His Ile Ser Thr Gly
1 5 10 15
Leu Ser Ile Phe Asp Thr Ser Leu Phe
20 25
<210>41
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<213> Artificial Sequence (Artificial Sequence)
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Gln Leu Gly Ser Leu Val Ser Asp Tyr Cys Asn Val Leu Asn Lys Glu
1 5 10 15
Phe Thr Ala Gly Ser Val Glu Ile Thr Leu Arg Ser Tyr Lys
20 2530
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<213> Artificial Sequence (Artificial Sequence)
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Ile Cys Lys Ala Phe Ile Asn Glu Ala Lys Ala His Gly Arg Glu Trp
1 5 10 15
Gly Gly Leu Met Ala Thr Leu Asn Ile Cys Asn Phe Trp Ala
20 25 30
<210>43
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<213> Artificial Sequence (Artificial Sequence)
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Ile Leu Arg Asn Asn Arg Val Arg Arg Arg Ala Glu Asn Ala Gly Asn
1 5 10 15
Asp Ala Cys Ser Ile Ala Cys Pro Ile Val Met Arg Tyr Val
20 25 30
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<213> Artificial Sequence (Artificial Sequence)
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Leu Asp His Leu Ile Val Val Thr Asp Arg Phe Phe Ile Gln Ala Pro
1 5 10 15
Ser Asn Arg Val Met Ile Pro Ala Thr Ile Gly Thr Ala Met
20 25 30
<210>45
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<213> Artificial Sequence (Artificial Sequence)
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Tyr Lys Leu Leu Lys His Ser Arg Val Arg Ala Tyr Thr Tyr Ser Lys
1 5 10 15
Val Leu Gly Val Asp Arg Ala Ala Ile Met Ala Ser Gly Lys
20 25 30
<210>46
<211>30
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>46
Gln Val Val Glu His Leu Asn Arg Met Glu Lys Glu Gly Leu Leu Ser
1 5 10 15
Ser Lys Phe Lys Ala Phe Cys Lys Trp Val Phe Thr Tyr Pro
20 25 30
<210>47
<211>30
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>47
Val Leu Glu Glu Met Phe Gln Thr Met Val Ser Ser Lys Thr Gly His
1 5 10 15
Leu Thr Asp Asp Val Lys Asp Val Arg Ala Leu Ile Lys Thr
20 25 30
<210>48
<211>30
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>48
Leu Pro Arg Ala Ser Tyr Ser Ser His Ala Gly Gln Arg Ser Tyr Val
1 5 10 15
Ser Gly Val Leu Pro Ala Cys Leu Leu Ser Thr Lys Ser Lys
20 25 30
<210>49
<211>30
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>49
Ala Val Glu Thr Pro Ile Leu Val Ser Gly Ala Asp Arg Met Asp Glu
1 5 10 15
Glu Leu Met Gly Asn Asp Gly Gly Ala Ser His Thr Glu Ala
20 25 30
<210>50
<211>30
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>50
Arg Tyr Ser Glu Ser Gly Gln Phe His Ala Phe Thr Asp Glu Leu Glu
1 5 10 15
Ser Leu Pro Ser Pro Thr Met Pro Leu Lys Pro Gly Ala Gln
20 25 30
<210>51
<211>30
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>51
Ser Ala Asp Cys Gly Asp Ser Ser Ser Ser Ser Ser Asp Ser Gly Asn
1 5 10 15
Ser Asp Thr Glu Gln Ser Glu Arg Glu Glu Ala Arg Ala Glu
20 25 30
<210>52
<211>30
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>52
Ala Pro Arg Leu Arg Ala Pro Lys Ser Arg Arg Thr Ser Arg Pro Asn
1 5 10 15
Arg Gly Gln Thr Pro Cys Pro Ser Asn Ala Glu Glu Pro Glu
20 25 30
<210>53
<211>30
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>53
Gln Pro Trp Ile Ala Ala Val His Gln Glu Ser Asp Glu Arg Pro Ile
1 5 10 15
Phe Pro His Pro Ser Lys Pro Thr Phe Leu Pro Pro Val Lys
20 25 30
<210>54
<211>30
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>54
Arg Lys Lys Gly Leu Arg Asp Ser Arg Glu Gly Met Phe Leu Pro Lys
1 5 10 15
Pro Glu Ala Gly Ser Ala Ile Ser Asp Val Phe Glu Gly Arg
20 25 30
<210>55
<211>30
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>55
Glu Val Cys Gln Pro Lys Arg Ile Arg Pro Phe His Pro Pro Gly Ser
1 5 10 15
Pro Trp Ala Asn Arg Pro Leu Pro Ala Ser Leu Ala Pro Thr
20 25 30
<210>56
<211>30
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>56
Pro Thr Gly Pro Val His Glu Pro Val Gly Ser Leu Thr Pro Ala Pro
1 5 10 15
Val Pro Arg Pro Leu Asp Pro Ala Pro Ala Val Thr Pro Glu
20 25 30
<210>57
<211>30
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>57
Ala Ser His Leu Leu Glu Asp Pro Asp Glu Glu Thr Ser Gln Ala Val
1 5 10 15
Lys Ala Leu Arg Glu Met Ala Asp Thr Val Ile Pro Gln Lys
20 25 30
<210>58
<211>30
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>58
Glu Glu Ala Ala Ile Cys Gly Gln Met Asp Leu Asn His Pro Pro Pro
1 5 10 15
Arg Gly His Leu Asp Glu Leu Thr Thr Thr Leu Glu Ser Met
20 25 30
<210>59
<211>30
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>59
Thr Glu Asp Leu Asn Leu Asp Ser Pro Leu Thr Pro Glu Leu Asn Glu
1 5 10 15
Ile Leu Asp Thr Phe Leu Asn Asp Glu Cys Leu Leu His Ala
20 25 30
<210>60
<211>15
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>60
Met His Ile Ser Thr Gly Leu Ser Ile Phe Asp Thr Ser Leu Phe
1 5 10 15
<210>61
<211>605
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>61
Met Arg Pro Lys Lys Asp Gly Leu Glu Asp Phe Leu Arg Leu Thr Pro
1 5 10 15
Glu Ile Lys Lys Gln Leu Gly Ser Leu Val Ser Asp Tyr Cys Asn Val
20 25 30
Leu Asn Lys Glu Phe Thr Ala Gly Ser Val Glu Ile Thr Leu Arg Ser
35 40 45
Tyr Lys Ile Cys Lys Ala Phe Ile Asn Glu Ala Lys Ala His Gly Arg
50 55 60
Glu Trp Gly Gly Leu Met Ala Thr Leu Asn Ile Cys Asn Phe Trp Ala
65 70 75 80
Ile Leu Arg Asn Asn Arg Val Arg Arg Arg Ala Glu Asn Ala Gly Asn
85 90 95
Asp Ala Cys Ser Ile Ala Cys Pro Ile Val Met Arg Tyr Val Leu Asp
100 105 110
His Leu Ile Val Val Thr Asp Arg Phe Phe Ile Gln Ala Pro Ser Asn
115 120 125
Arg Val Met Ile Pro Ala Thr Ile Gly Thr Ala Met Tyr Lys Leu Leu
130 135 140
Lys His Ser Arg Val Arg Ala Tyr Thr Tyr Ser Lys Val Leu Gly Val
145 150 155 160
Asp Arg Ala Ala Ile Met Ala Ser Gly Lys Gln Val Val Glu His Leu
165 170 175
Asn Arg Met Glu Lys Glu Gly Leu Leu Ser Ser Lys Phe Lys Ala Phe
180 185 190
Cys Lys Trp Val Phe Thr Tyr Pro Val Leu Glu Glu Met Phe Gln Thr
195 200 205
Met Val Ser Ser Lys Thr Gly His Leu Thr Asp Asp Val Lys Asp Val
210 215 220
Arg Ala Leu Ile Lys Thr Leu Pro Arg Ala Ser Tyr Ser Ser His Ala
225 230 235 240
Gly Gln Arg Ser Tyr Val Ser Gly Val Leu Pro Ala Cys Leu Leu Ser
245 250 255
Thr Lys Ser Lys Ala Val Glu Thr Pro Ile Leu Val Ser Gly Ala Asp
260 265 270
Arg Met Asp Glu Glu Leu Met Gly Asn Asp Gly Gly Ala Ser His Thr
275 280 285
Glu Ala Arg Tyr Ser Glu Ser Gly Gln Phe His Ala Phe Thr Asp Glu
290 295 300
Leu Glu Ser Leu Pro Ser Pro Thr Met Pro Leu Lys Pro Gly Ala Gln
305 310 315 320
Ser Ala Asp Cys Gly Asp Ser Ser Ser Ser Ser Ser Asp Ser Gly Asn
325 330 335
Ser Asp Thr Glu Gln Ser Glu Arg Glu Glu Ala Arg Ala Glu Ala Pro
340 345 350
Arg Leu Arg Ala Pro Lys Ser Arg Arg Thr Ser Arg Pro Asn Arg Gly
355 360 365
Gln Thr Pro Cys Pro Ser Asn Ala Glu Glu Pro Glu Gln Pro Trp Ile
370 375 380
Ala Ala Val His Gln Glu Ser Asp Glu Arg Pro Ile Phe Pro His Pro
385 390 395 400
Ser Lys Pro Thr Phe Leu Pro Pro Val Lys Arg Lys Lys Gly Leu Arg
405 410 415
Asp Ser Arg Glu Gly Met Phe Leu Pro Lys Pro Glu Ala Gly Ser Ala
420 425 430
Ile Ser Asp Val Phe Glu Gly Arg Glu Val Cys Gln Pro Lys Arg Ile
435 440 445
Arg Pro Phe His Pro Pro Gly Ser Pro Trp Ala Asn Arg Pro Leu Pro
450 455 460
Ala Ser Leu Ala Pro Thr Pro Thr Gly Pro Val His Glu Pro Val Gly
465 470 475 480
Ser Leu Thr Pro Ala Pro Val Pro Arg Pro Leu Asp Pro Ala Pro Ala
485 490 495
Val Thr Pro Glu Ala Ser His Leu Leu Glu Asp Pro Asp Glu Glu Thr
500 505 510
Ser Gln Ala Val Lys Ala Leu Arg Glu Met Ala Asp Thr Val Ile Pro
515 520 525
Gln Lys Glu Glu Ala Ala Ile Cys Gly Gln Met Asp Leu Asn His Pro
530 535 540
Pro Pro Arg Gly His Leu Asp Glu Leu Thr Thr Thr Leu Glu Ser Met
545 550 555 560
Thr Glu Asp Leu Asn Leu Asp Ser Pro Leu Thr Pro Glu Leu Asn Glu
565 570 575
Ile Leu Asp Thr Phe Leu Asn Asp Glu Cys Leu Leu His Ala Met His
580 585 590
Ile Ser Thr Gly Leu Ser Ile Phe Asp Thr Ser Leu Phe
595 600 605
<210>62
<211>511
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>62
Ser Ser Asp Ser Gly Asn Ser Asp Thr Glu Gln Ser Glu Arg Glu Glu
1 5 10 15
Ala Arg Ala Glu Ala Pro Arg Leu Arg Ala Pro Lys Ser Arg Glu Thr
20 25 30
Ser Gln Ala Val Lys Ala Leu Arg Glu Met Ala Asp Thr Val Ile Pro
3540 45
Gln Lys Glu Ala Ala Ile Cys Gly Gln Met Asp Glu Thr Ser Gln Ala
50 55 60
Val Lys Ala Leu Arg Glu Met Ala Asp Thr Val Ile Pro Gln Lys Glu
65 70 75 80
Ala Ala Ile Cys Gly Gln Met Asp Asp Glu Leu Thr Thr Thr Leu Glu
85 90 95
Ser Met Thr Glu Asp Leu Asn Leu Asp Ser Pro Leu Thr Pro Glu Leu
100 105 110
Asn Glu Ile Leu Asp Thr Asp Glu Leu Thr Thr Thr Leu Glu Ser Met
115 120 125
Thr Glu Asp Leu Asn Leu Asp Ser Pro Leu Thr Pro Glu Leu Asn Glu
130 135 140
Ile Leu Asp Thr Phe Leu Asn Asp Glu Cys Leu Leu His Ala Met His
145 150 155 160
Ile Ser Thr Gly Leu Ser Ile Phe Asp Thr Ser Leu Phe Pro Thr Gly
165 170 175
Pro Val His Glu Pro Val Gly Ser Leu Thr Pro Ala Pro Val Pro Arg
180 185 190
Pro Leu Asp Pro Ala Pro Ala Val Thr Pro Glu Pro Thr Gly Pro Val
195200 205
His Glu Pro Val Gly Ser Leu Thr Pro Ala Pro Val Pro Arg Pro Leu
210 215 220
Asp Pro Ala Pro Ala Val Thr Pro Glu Pro Thr Gly Pro Val His Glu
225 230 235 240
Pro Val Gly Ser Leu Thr Pro Ala Pro Val Pro Arg Pro Leu Asp Pro
245 250 255
Ala Pro Ala Val Thr Pro Glu His His His His His His Met Ser Pro
260 265 270
Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro Thr Arg Leu
275 280 285
Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu Tyr Glu Arg
290 295 300
Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu Gly Leu Glu
305 310 315 320
Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys Leu Thr Gln
325 330 335
Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn Met Leu Gly
340 345 350
Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu Gly Ala Val
355 360365
Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser Lys Asp Phe
370 375 380
Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu Met Leu Lys
385 390 395 400
Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn Gly Asp His
405 410 415
Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp Val Val Leu
420 425 430
Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu Val Cys Phe
435 440 445
Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr Leu Lys Ser
450 455 460
Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala Thr Phe Gly
465 470 475 480
Gly Gly Asp His Pro Pro Lys Ser Asp Leu Glu Val Leu Phe Gln Gly
485 490 495
Pro Leu Gly Ser Pro Glu Phe Pro Gly Arg Leu Glu Arg Pro His
500 505 510
<210>63
<211>511
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>63
Ser Ser Asp Ser Gly Asn Ser Asp Thr Glu Gln Ser Glu Arg Glu Glu
1 5 10 15
Ala Arg Ala Glu Ala Pro Arg Leu Arg Ala Pro Lys Ser Arg Glu Thr
20 25 30
Ser Gln Ala Val Lys Ala Leu Arg Glu Met Ala Asp Thr Val Ile Pro
35 40 45
Gln Lys Glu Ala Ala Ile Cys Gly Gln Met Asp Asp Glu Leu Thr Thr
50 55 60
Thr Leu Glu Ser Met Thr Glu Asp Leu Asn Leu Asp Ser Pro Leu Thr
65 70 75 80
Pro Glu Leu Asn Glu Ile Leu Asp Thr Phe Leu Asn Asp Glu Cys Leu
85 90 95
Leu His Ala Met His Ile Ser Thr Gly Leu Ser Ile Phe Asp Thr Ser
100 105 110
Leu Phe Pro Thr Gly Pro Val His Glu Pro Val Gly Ser Leu Thr Pro
115 120 125
Ala Pro Val Pro Arg Pro Leu Asp Pro Ala Pro Ala Val Thr Pro Glu
130 135 140
Glu Thr Ser Gln Ala Val Lys Ala Leu Arg Glu Met AlaAsp Thr Val
145 150 155 160
Ile Pro Gln Lys Glu Ala Ala Ile Cys Gly Gln Met Asp Asp Glu Leu
165 170 175
Thr Thr Thr Leu Glu Ser Met Thr Glu Asp Leu Asn Leu Asp Ser Pro
180 185 190
Leu Thr Pro Glu Leu Asn Glu Ile Leu Asp Thr Pro Thr Gly Pro Val
195 200 205
His Glu Pro Val Gly Ser Leu Thr Pro Ala Pro Val Pro Arg Pro Leu
210 215 220
Asp Pro Ala Pro Ala Val Thr Pro Glu Pro Thr Gly Pro Val His Glu
225 230 235 240
Pro Val Gly Ser Leu Thr Pro Ala Pro Val Pro Arg Pro Leu Asp Pro
245 250 255
Ala Pro Ala Val Thr Pro Glu His His His His His His Met Ser Pro
260 265 270
Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro Thr Arg Leu
275 280 285
Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu Tyr Glu Arg
290 295 300
Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu Gly LeuGlu
305 310 315 320
Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys Leu Thr Gln
325 330 335
Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn Met Leu Gly
340 345 350
Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu Gly Ala Val
355 360 365
Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser Lys Asp Phe
370 375 380
Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu Met Leu Lys
385 390 395 400
Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn Gly Asp His
405 410 415
Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp Val Val Leu
420 425 430
Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu Val Cys Phe
435 440 445
Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr Leu Lys Ser
450 455 460
Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala Thr Phe Gly
465 470 475 480
Gly Gly Asp His Pro Pro Lys Ser Asp Leu Glu Val Leu Phe Gln Gly
485 490 495
Pro Leu Gly Ser Pro Glu Phe Pro Gly Arg Leu Glu Arg Pro His
500 505 510
<210>64
<211>392
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>64
Ser Ser Asp Ser Gly Asn Ser Asp Thr Glu Gln Ser Glu Arg Glu Glu
1 5 10 15
Ala Arg Ala Glu Ala Pro Arg Leu Arg Ala Pro Lys Ser Arg Glu Thr
20 25 30
Ser Gln Ala Val Lys Ala Leu Arg Glu Met Ala Asp Thr Val Ile Pro
35 40 45
Gln Lys Glu Ala Ala Ile Cys Gly Gln Met Asp Asp Glu Leu Thr Thr
50 55 60
Thr Leu Glu Ser Met Thr Glu Asp Leu Asn Leu Asp Ser Pro Leu Thr
65 70 75 80
Pro Glu Leu Asn Glu Ile Leu Asp Thr Phe Leu Asn Asp Glu Cys Leu
85 9095
Leu His Ala Met His Ile Ser Thr Gly Leu Ser Ile Phe Asp Thr Ser
100 105 110
Leu Phe Pro Thr Gly Pro Val His Glu Pro Val Gly Ser Leu Thr Pro
115 120 125
Ala Pro Val Pro Arg Pro Leu Asp Pro Ala Pro Ala Val Thr Pro Glu
130 135 140
His His His His His His Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile
145 150 155 160
Lys Gly Leu Val Gln Pro Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu
165 170 175
Lys Tyr Glu Glu His Leu Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg
180 185 190
Asn Lys Lys Phe Glu Leu Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr
195 200 205
Ile Asp Gly Asp Val Lys Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr
210 215 220
Ile Ala Asp Lys His Asn Met Leu Gly Gly Cys Pro Lys Glu Arg Ala
225 230 235 240
Glu Ile Ser Met Leu Glu Gly Ala Val Leu Asp Ile Arg Tyr Gly Val
245 250 255
Ser Arg Ile Ala Tyr Ser Lys Asp Phe Glu Thr Leu Lys Val Asp Phe
260 265 270
Leu Ser Lys Leu Pro Glu Met Leu Lys Met Phe Glu Asp Arg Leu Cys
275 280 285
His Lys Thr Tyr Leu Asn Gly Asp His Val Thr His Pro Asp Phe Met
290 295 300
Leu Tyr Asp Ala Leu Asp Val Val Leu Tyr Met Asp Pro Met Cys Leu
305 310 315 320
Asp Ala Phe Pro Lys Leu Val Cys Phe Lys Lys Arg Ile Glu Ala Ile
325 330 335
Pro Gln Ile Asp Lys Tyr Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro
340 345 350
Leu Gln Gly Trp Gln Ala Thr Phe Gly Gly Gly Asp His Pro Pro Lys
355 360 365
Ser Asp Leu Glu Val Leu Phe Gln Gly Pro Leu Gly Ser Pro Glu Phe
370 375 380
Pro Gly Arg Leu Glu Arg Pro His
385 390
Claims (8)
1. The recombinant Rta protein for detecting nasopharyngeal carcinoma is characterized in that the amino acid sequence of the recombinant Rta protein is represented by a sequence 12, a sequence 18, a sequence 39, a sequence 40 and a sequence 56 in a sequence table according to the sequence 1: 2: 2: 1: 3, and the amino acid sequence is a sequence 62 or a sequence 63 in a sequence table.
2. A gene encoding the recombinant Rta protein of claim 1.
3. An expression cassette, vector or cell comprising the gene of claim 2.
4. The method for producing a recombinant Rta protein according to claim 1, comprising the steps of: synthesizing the coding gene of the recombinant Rta protein, constructing an expression vector, transforming a protein expression host, and performing protein expression and purification.
5. The polypeptide composition for detecting nasopharyngeal carcinoma is characterized in that the polypeptide with amino acid sequences of sequence 12, sequence 18, sequence 39, sequence 40 and sequence 56 in a sequence table is prepared according to the sequence number 1: 2: 2: 1: 3 in a molar ratio.
6. The method for preparing the polypeptide composition of claim 5, which comprises synthesizing the polypeptides represented by sequence 12, sequence 18, sequence 39, sequence 40 and sequence 56 in the sequence table respectively and performing the steps according to 1: 2: 2: 1: 3, and mixing uniformly.
7. A reagent or kit comprising the recombinant Rta protein of claim 1 or the polypeptide composition of claim 5.
8. The reagent or kit of claim 7, wherein the working concentration of the recombinant Rta protein or the polypeptide composition is 1.0 μ g/ml.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911281000.2A CN110698545B (en) | 2019-12-13 | 2019-12-13 | Recombinant Rta protein for nasopharyngeal carcinoma detection, kit and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201911281000.2A CN110698545B (en) | 2019-12-13 | 2019-12-13 | Recombinant Rta protein for nasopharyngeal carcinoma detection, kit and application thereof |
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| CN110698545B true CN110698545B (en) | 2020-04-07 |
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| CN101153059B (en) * | 2006-09-27 | 2010-05-12 | 同昕生物技术（北京）有限公司 | ELISA reagent kit for screening, diagnosis and treatment effect forecast of nasopharyngeal carcinoma |
| CN105044355B (en) * | 2015-07-08 | 2016-10-19 | 同昕生物技术（北京）有限公司 | For detecting chemical luminescence reagent kit and the application thereof of Epstein-Barr virus Rta/IgG antibody |
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