CN1584593A - Reagent box for enzyme linked immunosorbent assay of EB virus protease and its preparation - Google Patents

Reagent box for enzyme linked immunosorbent assay of EB virus protease and its preparation Download PDF

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CN1584593A
CN1584593A CN 03154287 CN03154287A CN1584593A CN 1584593 A CN1584593 A CN 1584593A CN 03154287 CN03154287 CN 03154287 CN 03154287 A CN03154287 A CN 03154287A CN 1584593 A CN1584593 A CN 1584593A
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albumen
epstein
vca
barr virus
preparation
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CN1300582C (en
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吴文翰
陈泓霖
陈国雄
吴子柏
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HONGKONG SHENNONG CO Ltd
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HONGKONG SHENNONG CO Ltd
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Priority to CN 200610077830 priority patent/CN1920562A/en
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Abstract

A method for preparing EB viral protein enzyme-linked immunosorbent diagnostic kit includes selectng EBNAl (BKRF1) prote, Zta (BZLF1) protein and VCA-p18 protin in EB viral protein for diagnosing nasopharyngeal carcinoma of serodiagnosis as target antigen for detecting antibody level in blood serum, using glutathione-transferase gene fusion system to carry on clone, presentation and purification of EB viral protein for creating diagnostic kit.

Description

Epstein-Barr virus enzyme linked immunological absorption diagnostic kit and preparation method thereof
[technical field]
The present invention relates to nasopharyngeal carcinoma and other Epstein-Barr virus relevant diseases are carried out serodiagnostic kit and preparation method thereof, particularly adopt specific Epstein-Barr virus albumen to detect the kit of antibody horizontal in the serum and the method that the Epstein-Barr virus recombinant polypeptide prepares enzyme-linked immunosorbent assay kit as target antigen.
[background technology]
In China, almost the crowd more than 100% 4 years old has all infected Epstein-Barr virus, and the health adult more than 80% is the Epstein-Barr virus carrier.Can exist a kind of in the Epstein-Barr virus carrier serum or 2 to 3 kinds of (not being multiple, promptly is not more than 3 kinds) low-level (not being high-caliber) anti-Epstein-Barr virus antibodies.China south, the particularly Delta of the Pearl River and area, Hong Kong be the district occurred frequently of nasopharyngeal carcinoma in the world, and the generation of nasopharyngeal carcinoma and ebv infection are closely related.China south and area, Hong Kong also are other Epstein-Barr virus relevant diseases, for example the district occurred frequently of the lymphoepithelioma sample cancer of the NK/T cell lymphoma of nasal cavity, nasal sinus, lung and infectious mononucleosis etc.The above two patients serum's Epstein-Barr virus antibody repertoires and nasopharyngeal carcinoma patient roughly the same, and infectious mononucleosis patients serum Epstein-Barr virus antibody have certain priority regularity.
The serum Epstein-Barr virus detection of antibodies of using clinically routinely (immunofluorescence technique or immune enzyme linked immunosorbent assay) is owed objectivity (calculating the percent of positive cell with the individual at microscopically) to a certain extent at present, index is thick (adopts the titre of geometric series, for example 1: 20,1: 40,1: 80 etc., do not adopt the very concrete numeral of present digital age as yet), and not strong to the feature specific aim of ebv infection.For example nasopharyngeal carcinoma mainly is that latent infection companion minimal amounts of dissolved sexuality is dyed, and has the high-caliber anti-Epstein-Barr virus antibody of wide spectrum, particularly IgA antibody among the nasopharyngeal carcinoma patients serum mostly.For example can not show the distinctive antibody horizontal of tool among the infectious mononucleosis patients serum (comprising: p18 VCA IgM, low compatibility p18 VCAIgG, p18 VCA IgG and EBNA1 IgG) again definitely.
Enzyme linked immunosorbent assay can be used for differentiating the various Infection Status of Epstein-Barr virus, when serodiagnosis Epstein-Barr virus relevant disease, has very potential advantage.Though the existing at present research report that adopts enzyme connection absorption trial-production box serodiagnosis Epstein-Barr virus relevant disease lacks a cover new method of handling the Epstein-Barr virus antibody horizontal.How to distinguish high and low level, still incomparably than objective standard.In addition, when using enzyme linked immunosorbent assay, how to overcome the difference between each lot number and adopt the same lot number kit difference between the detection at every turn, still do not have good recipe at present.
[summary of the invention]
The objective of the invention is to overcome the deficiencies in the prior art part, a kind of Epstein-Barr virus enzyme linked immunological absorption diagnostic kit is provided, the enzyme linked immunosorbent assay of its optimization (ELISA) has the specificity of objectivity and Epstein-Barr virus antigen, and it provides the hypotype of detection by quantitative at the different immune globulin antibodies of individual Epstein-Barr virus antigen.The enzyme linked immunosorbent assay of this optimization (ELISA) is new fully, is that traditional serological detection method is not accomplished, because the specificity that traditional serological detection method can not clear and definite antigen.And the enzyme linked immunosorbent assay of new optimization (ELISA) has adopted reference serum, can correct the difference between every batch of detection, has guaranteed the reliability of diagnosis.
The Epstein-Barr virus albumen (homology of related amino acid sequence 95%) that the present invention selects the tool diagnostic value of serodiagnosis nasopharyngeal carcinoma (or infectious mononucleosis) is back one section of EBNA1 (BKRF1) albumen, that is:
PVGEADYFEYHQEGGPDGEPDVPPGAIEQGPADDPGEGPSTGP
RGQGDGGRRKKGGWFGKHRGQGGSNPKFENIAEGLRALLARSHVERTTDEGTWVAGVF
VYGGSKTSLYNLRRGTALAIPQCRLTPLSRLPFGMAPGPGPQPGPLRESIVCYFMVFL
QTHIFAEVLKDAIKDLVMTKPAPTCNIRVTVCSFDDGVDLPPWFPPMVEGAAAEGDDG
DDGDEGGDGDEGEEGQE
And Zta (BZLF1) albumen and VCA-p18 albumen (being VCA or the BFRF3 albumen of 18Kd).Adopt these three kinds of Epstein-Barr virus albumen as target antigen, in order to detect the antibody horizontal in the serum, and by in glutathione transferase gene fusion system to Epstein-Barr virus albumen clone, expression and purifying, formulate out diagnostic kit, thereby recombined EB virus antigen used aspect the following diagnosis:
1, the primary infection of Epstein-Barr virus;
2, establish the antibody repertoire and the various ebv infections of antidiastole of ebv infection;
3, differentiate infectious mononucleosis (Infectious mononucleosis) and infectiousness monocyte syndrome (Infectious mononucleosissyndrome);
4, serology is assisted to diagnose to doubt and is nasopharyngeal carcinoma;
5, the risk of nasopharyngeal carcinoma is suffered from early detection and assessment.
The kit of these three kinds of albumen (six kinds of antibody tests) can be complementary mutually.May be used alone, can also be used in combination.For example: the combine detection of EBNA1-IgA and Zta-IgG has improved the reliability of serodiagnosis nasopharyngeal carcinoma widely.That is: most nasopharyngeal carcinoma patients both had been the EBNA1-IgA positive, were the Zta-IgG positive again; Even minority nasopharyngeal carcinoma patient EBNA1-IgA feminine gender also can be the Zta-IgG positive.Even and minority nasopharyngeal carcinoma patient Zta-IgG feminine gender also can be the EBNA1-IgA positive, both have complementation.
Many people that are suspected to have nasopharyngeal carcinoma clinically, after this combine detection, if EBNA1-IgA and Zta-IgG are all negative, then can get rid of more than 90% is the nasopharyngeal carcinoma patient surely.This contributes much concerning the hospital and crowd of nasopharyngeal carcinoma district occurred frequently undoubtedly.
Does is early detection patient in the crowd of nasopharyngeal carcinoma district occurred frequently how so that early treatment obtain best prognosis? being used in combination of this kit provides possibility.Show after being EBNA1-IgA, EBNA1-IgG and Zta-IgG simultaneously: three's positive is the excessive risk crowd; Both positives are middle risk population; Only Dan Yangzhe is the low-risk crowd.This is again a major contribution to nasopharyngeal carcinoma district occurred frequently crowd's early detection nasopharyngeal carcinoma patient.
Being used in combination this kit also helps the diagnosis of infectious mononucleosis and differentiates various types of ebv infections.
In a word; select the target antigen of these three kinds of Epstein-Barr virus albumen as kit; the value that independent use is not only arranged; a globality, that have complementarity especially design and clinical practice (seeing the following form); can be used in combination; non-other kits can be compared, and have novelty, should obtain rights protection.Please see the following form.
Enzyme linked immunosorbent assay is applied to
The serodiagnosis of EBNA1-IgA nasopharyngeal carcinoma
The serodiagnosis of EBNA1-IgG nasopharyngeal carcinoma and infectious mononucleosis
The serodiagnosis of Zta-IgA nasopharyngeal carcinoma
The serodiagnosis of Zta-IgG nasopharyngeal carcinoma
The serodiagnosis of P18 VCA IgG infectious mononucleosis
The serodiagnosis of P18 VCA IgM infectious mononucleosis acute stage
The serodiagnosis of P18 VCA IgG+EBNA1-IgG Epstein-Barr virus recent infection
The serodiagnosis of EBNA1-IgA+Zta-IgG nasopharyngeal carcinoma
Or
EBNA1-IgA+Zta-IgA
EBNA1-IgA+EBNA1-IgG+Zta-IgG suffers from the risk assessment of nasopharyngeal carcinoma
Utilize these three kinds of Epstein-Barr virus albumen of the glutathione transferase gene fusion clone of system, expression and purifying.The size of fusion cloning albumen is respectively:
EBNA-1＝40Kd；BFRF3＝44Kd；BZLF1＝53Kd。
But being coated on the antigen on the check-out console, having adopted fibrin ferment (Thrombin) method to break away from from fusion, is the antigen of having removed glutathione transferase (GST) therefore.This has just avoided when antigen-antibody reaction by the caused nonspecific reaction of glutathione transferase.
The feature of these three kinds of Epstein-Barr virus albumen can be summarized as follows table:
Epstein-Barr virus molecular weight of albumen (amino acid no) Epstein-Barr virus gene nucleotide (quite coordinate)
EBNA1 40Kd(234) BKRF1 705(109171-109875)
Zta 53Kd(245) BZLF1 738(102210-103155)
P18VCA 44Kd(176) BFRF3 531(61507-62037)
[description of drawings]
Fig. 1: the dilutability of serum when adopting ELISA to detect the EBNA1-IgA antibody horizontal.
Fig. 2: the BNA1-IgA antibody horizontal of nasopharyngeal carcinoma patient and healthy relatively.
Fig. 3: the Epstein-Barr virus ELISA method of optimizing the in-vitro diagnosis nasopharyngeal carcinoma.
Fig. 4: the antibody that has high-level wide spectrum in the serum is nasopharyngeal carcinoma patient's a species specific feature.
Fig. 5: the Epstein-Barr virus antibody repertoire of nasopharyngeal carcinoma diagnosis and risk assessment.
[embodiment]
One, Epstein-Barr virus enzyme linked immunological absorption diagnostic kit, include: positive control serum, control serum, zymolyte, cessation reaction liquid, dilution buffer liquid, dcq buffer liquid and Epstein-Barr virus target antigen, wherein Epstein-Barr virus albumen is: back one section of EBNA1 (BKRF1) albumen, Zta (BZLF1) albumen and VCA-p18 albumen (being VCA or the BFRF3 albumen of 18Kd).
Kit also includes the diagnosis reference serum that is equivalent to optimize critical optical density value.Reference serum is best critical value with the relative optical density value (rOD) of sensitivity curve and specificity curve interface point.
The preparation method of Epstein-Barr virus enzyme linked immunological absorption diagnostic kit, the preparation, purifying mark, the titration of tiring, bag that comprises recombinant antigen is by plate preparation, sealing, drying and packing, the preparation of enzyme labelled antibody concentrate, dilution preparation, positive control preparation, normal control preparation, reference serum preparation, substrate packing, stop buffer and concentrate step such as purchase.
Two, clone, expression and the purifying of Epstein-Barr virus albumen in glutathione transferase gene fusion system
2.1 select the Epstein-Barr virus albumen of serodiagnosis nasopharyngeal carcinoma (or infectious mononucleosis)
The Epstein-Barr virus albumen of tool diagnostic value is: back one section of EBNA1 (BKRF1) albumen, Zta (BZLF1) albumen and VCA-p18 albumen (being VCA or the BFRF3 albumen of 18Kd).Therefore we adopt these three kinds of Epstein-Barr virus albumen as target antigen, in order to detect the antibody horizontal in the serum.
2.2 the preparation of three kinds of Epstein-Barr virus albumen
Adopt modern molecular biology method these three kinds of Epstein-Barr virus albumen of clone, expression and purifying in glutathione transferase gene fusion system.
2.2.1 the selected mRNA sequence of these three kinds of Epstein-Barr virus albumen in gene pool (V01555, the sequence of B95-8 cell line), reverse transcriptase to the cDNA sequence of transferring.
The primer of this cDNA sequence 2.2.2 design can be increased
Add the BamHI enzyme at primer 5 ' end and cut sequence (GGATCC) or EcoRI sequence (GAATTC), so that after Bam HI and EcoR1 cutting, it embolus is linked to plasmid pGEX DNA (4948bp, Amersham Biosciences).And primer 5 ' end adds TAG again, and both favourable insertion does not appear among the clone after Restriction Enzyme digestion again.
2.2.3 concrete design of primers, sequence amplification process
2.2.3.1 about Epstein-Barr virus EBNA1 albumen (claiming BKRF1 albumen again)
2.2.3.1.1 EBNA-1 (BKRF1) design of primers
5’-TAC? GGA?TCC?CCT?GTA?GGG?GAA?GCC?GAT?TA-3’
BamHI
5’-TAC? GAA?TTC?TCA?CTC?CTG?CCC?TTC?CTC?CAC-3’
EcoRI
3 base TAC of primer 5 ' end do not appear among the clone after Restriction Enzyme digestion.
GGATCC is BamHI restriction enzyme digestion sequence (Bam HI restriction site),
GAATTC then is EcoRI restriction enzyme digestion sequence (Bam HI restriction site).
2.2.3.1.2 the EBNA-1 coded sequence (cDNA) of amplification
The nucleotide coding sequence of the EBNA1 of B95-8 cell (BKRF1) should be 107950-109875.The sequence of amplification is 109171-109875 now.Promptly need not the EBNA-1 coded sequence of 107950-109170 glycocoll (Gly) duplicate block intercepting.DNA sequences encoding of being cloned and B95-8 cell in full accord, totally 705 bases.
Represent with underscore.
107950→a?tgtctgacga?ggggccaggt?acaggacctg?gaaatggcct?aggagagaag
108061?aaccatggac?gaggacgggg?aagaggacga?ggacgaggag?gcggaagacc?aggagccccg
108121?ggcggctcag?gatcagggcc?aagacataga?gatggtgtcc?ggagacccca?aaaacgtcca
108001?ggagacacat?ctggaccaga?aggctccggc?ggcagtggac?ctcaaagaag?agggggtgat
108061 aaccatggac?gaggacgggg?aagaggacga ggacgaggag?gcggaagacc?aggagccccg
108121 ggcggctcag?gatcagggcc?aagacataga gatggtgtcc?ggagacccca?aaaacgtcca
108181 agttgcattg?gctgcaaagg?gacccacggt ggaacaggag?caggagcagg?agcgggaggg
108241 gcaggagcag?gaggggcagg?agcaggagga ggggcaggag?caggaggagg?ggcaggaggg
108301 gcaggagggg?caggaggggc?aggagcagga ggaggggcag?gagcaggagg?aggggcagga
108361 ggggcaggag?gggcaggagc?aggaggaggg gcaggagcag?gaggaggggc?aggaggggca
108421 ggagcaggag?gaggggcagg?aggggcagga ggggcaggag?caggaggagg?ggcaggagca
108481 ggaggagggg?caggaggggc?aggagcagga ggaggggcag?gaggggcagg?aggggcagga
108541 gcaggaggag?gggcaggagc?aggaggggca ggaggggcag?gaggggcagg?agcaggaggg
108601 gcaggagcag?gaggaggggc?aggaggggca ggaggggcag?gagcaggagg?ggcaggagca
108661 ggaggggcag?gagcaggagg?ggcaggagca ggaggggcag?gaggggcagg?agcaggaggg
108721 gcaggagggg?caggagcagg?aggggcagga ggggcaggag?caggaggagg?ggcaggaggg
108781 gcaggagcag?gaggaggggc?aggaggggca ggagcaggag?gggcaggagg?ggcaggagca
108841 ggaggggcag?gaggggcagg?agcaggaggg gcaggagggg?caggagcagg?aggaggggca
108901 ggagcaggag?gggcaggagc?aggaggtgga ggccggggtc?gaggaggcag?tggaggccgg
108961 ggtcgaggag?gtagtggagg?ccggggtcga ggaggtagtg?gaggccgccg?gggtagagga
109021 cgtgaaagag?ccaggggggg?aagtcgtgaa agagccaggg?ggagaggtcg?tggacgtgga
109081 gaaaagaggc?ccaggagtcc?cagtagtcag tcatcatcat?ccgggtctcc?accgcgcagg
109171
↓
109141 ccccctccag?gtagaaggcc?atttttccac? cctgtagggg?aagccgatta?ttttgaatac
109201? caccaagaag?gtggcccaga?tggtgagcct gacgtgcccc?cgggagcgat?agagcagggc
109261? cccgcagatg?acccaggaga?aggcccaagc actggacccc?ggggtcaggg?tgatgggggc
109321? aggcgcaaaa?aaggagggtg?gtttggaaag catcgtggtc?aaggaggttc?caacccgaaa
109381? tttgagaaca?ttgcagaagg?tttaagagct ctcctggcta?ggagtcacgt?agaaaggact
109441? accgacgaag?gaacttgggt?cgccggtgtg ttcgtatatg?gaggtagtaa?gacctccctt
109501? tacaacctaa?ggcgaggaac?tgcccttgct attccacaat?gtcgtcttac?accattgagt
109561? cgtctcccct?ttggaatggc?ccctggaccc ggcccacaac?ctggcccgct?aagggagtcc
109621? attgtctgtt?atttcatggt?ctttttacaa actcatatat?ttgctgaggt?tttgaaggat
109681? gcgattaagg?accttgttat?gacaaagccc gctcctacct?gcaatatcag?ggtgactgtg
109741? tgcagctttg?acgatggagt?agatttgcct ccctggtttc?cacctatggt?ggaaggggct
109801? gccgcggagg?gtgatgacgg?agatgacgga gatgaaggag?gtgatggaga?tgagggtgag
109861? gaagggcagg?agtga←109875
*It is terminator codon
2.2.3.1.3 the amino acid sequence (amino acid sequenc) that forms of translating
Except that the tga of 109873-109875 is that actual translations arrives the terminator codon
234 amino acid are represented with underscore.
MSDEGPGTGPGNGLGEKGDTSGPEGSGGSGPQRRGGDNHGRGRG
RGRGRGGGRPGAPGGSGSGPRHRDGVRRPQKRPSCIGCKGTHGGTGAGAGAGGAGAGG
AGAGGGAGAGGGAGGAGGAGGAGAGGGAGAGGGAGGAGGAGAGGGAGAGGGAGGAGAG
GGAGGAGGAGAGGGAGAGGGAGGAGAGGGAGGAGGAGAGGGAGAGGAGGAGGAGAGGA
GAGGGAGGAGGAGAGGAGAGGAGAGGAGAGGAGGAGAGGAGGAGAGGAGGAGAGGGAG
GAGAGGGAGGAGAGGAGGAGAGGAGGAGAGGAGGAGAGGGAGAGGAGAGGGGRGRGGS
GGRGRGGSGGRGRGGSGGRRGRGRERARGGSRERARGRGRGRGEKRPRSPSSQSSSSG
SPPRRPPPGRRPFFH PVGEADYFEYHQEGGPDGEPDVPPGAIEQGPADDPGEGPSTGP
RGQGDGGRRKKGGWFGKHRGQGGSNPKFENIAEGLRALLARSHVERTTDEGTWVAGVF
VYGGSKTSLYNLRRGTALAIPQCRLTPLSRLPFGMAPGPGPQPGPLRESIVCYFMVFL
QTHIFAEVLKDAIKDLVMTKPAPTCNIRVTVCSFDDGVDLPPWFPPMVEGAAAEGDDG
DDGDEGGDGDEGEEGQE
2.2.3.2 about Epstein-Barr virus Zta (BamH1 Z tRans aCtivator) or ZEBRA (BamHI Z EPstein- BArr REplication ACtivator) albumen or BZLF1 albumen
2.2.3.2.1 Zta (BZLF1) design of primers
5’-TAC? GGA?TCC?ATG?ATG?GAC?CCA?AAC?TCG?AC-3’
BamHI
5’-TAC? GAA?TTC?TTA?GAA?ATT?TAA?GAG?ATC?CTC-3’
EcoRI
3 base TAC of primer 5 ' end do not appear among the clone after Restriction Enzyme digestion.
GGATCC is BamHI restriction enzyme digestion sequence (Bam HI restriction site),
GAATTC then is EcoRI restriction enzyme digestion sequence (Bam HI restrictionsite).
2.2.3.2.2 the Zta coded sequence (cDNA) of amplification
The nucleotide coding sequence of the Zta of B95-8 cell (BZLF1) should be 102210-102338,102423-102530, and 102655-103155 is spliced for three sections.Represent with underscore.Three sections sequences that are spliced of DNA sequences encoding of being cloned and B95-8 cell are in full accord, totally 738 bases.Represent with underscore.
102210
↓
102181 tgaagcaggc?gtggtttcaa?taacgggag t?tagaaattta?agagatcctc?gtgtaaaaca
102241? tctggtgtcc?gggggataat?ggagtcaaca?tccaggcttg?ggcacatctg?cttcaacagg
102301? aggcgcagcc?tgtcattttc?agatgatttg?gcagcagcca?cctgcggaca?aaaatcaggc
↑
102338
102361 gtttagatgg?ggcatttatg?tttgggacgc?tagccgcctg?ggcattcgtg?ttagtatata
102423
↓
102421 ct gacctcac?ggtagtgctg?cagcagttgc?ttaaacttgg?cccggcattt?tctggaagcc
102481? acccgattct?tgtatcgctt?tatttctagt?tcagaatcgc?attcctccag?ctgcgagcaa
↑
102530
102541 gggaatgcgt?tactacaagt?ggtgcctagt?cagttgaaac?aagccccacc?atccgctgcc
102655
↓
102601 gcccctccat?gagccccacc?gtccgctgcc?gcccctcctt?gagcccctcc?ttac cgattc
102661? tggctgttgt?ggtttccgtg?tgcgtcgtgc?cggggcagcc?actggtgcag?gctgtggaac
102721? accaatgtct?gctagctgtt?gtccttggtt?agccccgggg?caagcaaaca?ccactgctgc
102781? tgctgtttga?acagtagaat?tgtctccagg?ttgaggtgct?tctcccccgg?cttggttagt
102841? ctgttgattc?tgggttatgt?cggagactgg?gaacagctga?ggtgctgcat?aagcttgata
102901? agcattctca?ggagcaggct?gaggggcaga?aaaccacgac?ccagtcggag?cggttgaaac
102961? atgataggca?gttagctggc?cttgtggcag?aggctctggc?agcaccggcc?acagcacaca
103021? aggcaaagga?gcttgcgatg?gccctcccag?gtcctgatag?actctggtag?cttggtcaaa
103081? agcttgtaca?aaaggcacct?ggtatgggtc?aggtgtaaat?tttacatctt?cagaagtcga
103141? gtttgggtcc?atcatcttca?gcaaagatag?caaaggtggc?cggcaaggtg?caatgtttag
↑
103155
Three sections sequences that are spliced are as follows:
*〉be terminator codon
t?tagaaattta?agagatcctc?gtgtaaaaca
tctggtgtcc?gggggataat?ggagtcaaca?tccaggcttg?ggcacatctg?cttcaacagg
aggcgcagcc?tgtcattttc?agatgatttg?gcagcagc
gacctcac?ggtagtgctg?cagcagttgc?ttaaacttgg?cccggcattt?tctggaagcc
acccgattct?tgtatcgctt?tatttctagt?tcagaatcgc?attcctccag
cgattc
tggctgttgt?ggtttccgtg?tgcgtcgtgc?cggggcagcc?actggtgcag?gctgtggaac
accaatgtct?gctagctgtt?gtccttggtt?agccccgggg?caagcaaaca?ccactgctgc
tgctgtttga?acagtagaat?tgtctccagg?ttgaggtgct?tctcccccgg?cttggttagt
ctgttgattc?tgggttatgt?cggagactgg?gaacagctga?ggtgctgcat?aagcttgata
agcattctca?ggagcaggct?gaggggcaga?aaaccacgac?ccagtcggag?cggttgaaac
atgataggca?gttagctggc?cttgtggcag?aggctctggc?agcaccggcc?acagcacaca
aggcaaagga?gcttgcgatg?gccctcccag?gtcctgatag?actctggtag?cttggtcaaa
agcttgtaca?aaaggcacct?ggtatgggtc?aggtgtaaat?tttacatctt?cagaagtcga
gtttgggtcc?atcat
2.2.3.2.3 the amino acid sequence (amino acid sequenc) that forms of translating
Taa except that 102212-102210) be the terminator codon, actual translations to 245 amino acid is represented with underscore.
MMDPNSTSEDVKFTPDPYQVPFVQAFDQATRVYQDLGGPSQAPL
PCVLWPVLPEPLPQGQLTAYHVSTAPTGSWFSAPQPAPENAYQAYAAPQLFPVSDITQ
NQQTNQAGGEAPQPGDNSTVQTAAAVVFACPGANQGQQLADIGVPQPAPVAAPARRTR
KPQQPESLEECDSELEIKRYKNRVASRKCRAKFKQLLQHYREVAAAKSSENDRLRLLL
KQMCPSLDVDSIIPRTPDVLHEDLLNF
2.2.3.3 about Epstein-Barr virus VCA-p18 albumen (claiming BFRF3 albumen again), i.e. the VCA albumen of 18kd
2.2.3.3.1 VCA-p18 (BFRF3) design of primers
5’-TAC? GGA?TCC?ATG?GCA?CGC?CGG?CTG?CCC?AAG-3’
BamHI
5’-TAC? GAA?TTC?CTA?CTG?TTT?CTT?ACG?TG-3’
EcoRI
3 base TAC of primer 5 ' end do not appear among the clone after Restriction Enzyme digestion.
GGATCC is BamHI restriction enzyme digestion sequence (Bam HI restriction site), and GAATTC then is EcoRI restriction enzyme digestion sequence (Bam HI restrictionsite).
2.2.3.3.2 the BFRF3 coded sequence (cDNA) of amplification
The nucleotide coding sequence of the BFRF3 of B95-8 cell should be 61507-62037.
DNA sequences encoding of being cloned and B95-8 cell in full accord, totally 531 bases.Represent with underscore.
61507
↓
61501 cgcgtt atgg?cacgccggct?gcccaagccc?accctccagg?ggaggctgga?ggcggatttt
61561? ccagacagtc?ccctgcttcc?taaatttcaa?gagctgaacc?agaataatct?ccccaatgat
61621? gtttttcggg?aggctcaaag?aagttacctg?gtatttctga?catcccagtt?ctgctacgaa
61681? gagtacgtgc?agaggacttt?tggggtgcct?cggcgccaac?gcgccataga?caagaggcag
61741? agagccagtg?tggctggggc?tggtgctcat?gcacaccttg?gcgggtcatc?cgccaccccc
61801? gtccagcagg?ctcaggccgc?cgcatccgct?gggaccgggg?ccttggcatc?atcagcgccg
61861? tccacggccg?tagcccagtc?cgcgaccccc?tctgtttctt?catctattag?cagcctccgg
61921? gccgcgactt?cgggggcgac?tgccgccgcc?tccgccgccg?cagccgtcga?taccgggtca
61981? ggtggcgggg?gacaacccca?cgacaccgcc?ccacgcgggg?cacgtaagaa?acagtagagg
↑
62037
61507→ atgg?cacgccggct?gcccaagccc?accctccagg?ggaggctgga?ggcggatttt
ccagacagtc?ccctgcttcc?taaatttcaa?gagctgaacc?agaataatct?ccccaatgat
gtttttcggg?aggctcaaag?aagttacctg?gtatttctga?catcccagtt?ctgctacgaa
gagtacgtgc?agaggacttt?tggggtgcct?cggcgccaac?gcgccataga?caagaggcag
agagccagtg?tggctggggc?tggtgctcat?gcacaccttg?gcgggtcatc?cgccaccccc
gtccagcagg?ctcaggccgc?cgcatccgct?gggaccgggg?ccttggcatc?atcagcgccg
tccacggccg?tagcccagtc?cgcgaccccc?tctgtttctt?catctattag?cagcctccgg
gccgcgactt?cgggggcgac?tgccgccgcc?tccgccgccg?cagccgtcga?taccgggtca
ggtggcgggg?gacaacccca?cgacaccgcc?ccacgcgggg?cacgtaagaa?acagtag←62037
*〉be terminator codon
2.2.3.3.3 the amino acid sequence (amino acid sequenc) that forms of translating
Except that the tag of 62035-620370 is the terminator codon, actual translations to 176 amino acid is represented with underscore.
MARRLPKPTLQGRLEADFPDSPLLPKFQELNQNNLPNDVFREAQ
RSYLVFLTSQFCYEEYVQRTFGVPRRQRAIDKRQRASVAGAGAHAHLGGSSATPVQQA
QAAASAGTGALASSAPSTAVAQSATPSVSSSISSLRAATSGATAAASAAAAVDTGSGG
GGQPHDTAPRGARKKQ
Summary is as follows about the amino acid no of genetic fragment, gene pool sequence size, amplification cloned sequence size and the express polypeptide of Epstein-Barr virus EBNA1 albumen, Zta albumen and VCA-p18 albumen.
Table 1
Epstein-Barr virus albumen EBNA1 Zta VCA-p18
Genetic fragment BKRF1 BZLF BFRF3
Base is with storehouse sequence size 1926bp 738bp 531bp
Amplification cloned sequence size 705bp 738bp 531bp
The amino acid no 234aa 245aa 176aa of express polypeptide
2.3 (Pharmacia, Uppsala are tied on Sweden) to the procaryotic cell expression pGEX2T plasmid vector of Eco Rl/Bam HI digestion with pcr amplification and glutathione transferase (GST) gene clone.Adopt the lime chloride technology that the plasmid of reorganization is gone to Escherichia coli.Add lactose analog isopropylthiogalactoside and induce the Escherichia coli a few hours that forwarded recombinant plasmid to, gather in the crops bacterium then and with de-sludging triton (trade names of a series of organic nonionic surfactants) X-100 and ultrasonic treatment bacterium.The bacterium pyrolysis product is removed fragment after centrifugal and is clarified, and the supernatant that contains fusion adopts glutathione agarose 4B through glutathione transferase-glutathione affinity chromatography instrument purifying.Purity reaches more than 94.0%.The size of fusion cloning albumen is: EBNA-1=40Kd; BFRF3=44Kd; BZLF1=53Kd.Concrete steps are as follows.
2.3.1.cDNA amplification
2.3.1.1 the preparation of RNA.
2.3.1.1.1 requirement isolation of RNA from cultured cell according to Rnaid Plus kit (BIO 101) instructions.
Adopt RNaid Plus kit extracting RNA from the cell that suspends.Cultured cells is washed once with PBS, and per then 10 6Cell adds 1ml quanidine thiocyanate lysate, and mixes with vortex.Add isometric(al) acidic phenol (acid phenol) again, and mix.Add lmlchloroform isoamyl alcohol (BDH) to solution, mix once more.Lysate is after incubating 15min on the ice cube, at 4 ℃ of rotations 10 down, 000xg 20min.The upper strata is transferred to one newly manages, use chloroform isoamyl alcohol (BDH) the rotation 2min of 1/2 volume again.The upper strata is transferred to one newly manages.Add 15ul RNAMATRIX, and at room temperature hatch 5min, idol adds mixing in hatching, and makes RNA absorption.The RNA/RNAMATRIX compound is 10, and 1min forms bead under the 000xg, washes twice in 500 μ l RNA washing lotions.Bead is suspended in 30-100 μ l Diethpyrocarbonate once more, and (DEPC is Sigma) in the treated water.RNA is at 55 ℃ of following elution 5min, and is suitable for spectral analysis.
2.3.1.1.2. adopt RT pcr amplification rna transcription thing.
2.3.1.2 after Bam HI and EcoR1 cutting, embolus is linked to plasmid pGEX DNA (4948bp, Amersham Biosciences).
Go to the XL1 compentent cell 2.3.2 will link product.
2.3.3 winning indivedual cloning, employing makes the plasmid apertureization.
2.3.4 the restriction enzyme digestion of aperture DNA.
2.3.5 from pGEX reorganization thing, screen expressing fusion protein.
2.3.6 SDS PAGE analyzes.
Be Western blot 2.3.7 adopt anti-glutathione transferase antibody.
2.3.8 large-scale purification fusion.
2.3.9 with fibrin ferment the glutathione transferase in the glutathione transferase fusion is removed if necessary.
3. the characteristics of selected three class Epstein-Barr virus antigens
The ELISA method of selected three class Epstein-Barr virus antigens is not only responsive, special, but also objective to measuring Epstein-Barr virus antibody.
The ELISA method of table 2 Epstein-Barr virus antigen is not only responsive, special, but also objective to measuring Epstein-Barr virus antibody
Serum ebv infection state VCAIgG VCAIgA ZtaIgG ZtaIgA EBNA1 EBNA1
IgG IgA
N11 does not infect baby＜200 undetermined undetermined undetermined＜1k undetermineds
N31 infectiousness monokaryon is thin＞3k＜100＜200＜20＜1k＜100
Born of the same parents' increase disease patient
Healthy Epstein-Barr virus＞the 3k of MIN＜100＜200＜20 3k feminine genders
The carrier
PG495 takes from nasopharyngeal carcinoma＞3k 1.6k＞3k 100＞6k 800
The patients serum storehouse
PA196 takes from nasopharyngeal carcinoma＞3k＞1.6k＞3k 400＞6k＞800
The patients serum storehouse
Average optical density value=the S/N=3 of the average optical density value/serum blank of the critical value=serum of surveying
In order to detect Zta IgA, the serum dilution of beginning is 1: 20; In order to detect p18 VCAIgA and EBNA1 IgA, the serum dilution of beginning is 1: 100; In order to detect p18 VCAIgG and Zta IgG, the serum dilution of beginning is 1: 200; In order to detect EBNA1 IgG, the serum dilution of beginning is 1: 1000.Titre=the be higher than maximum serum dilution of critical value.
As known from Table 2:
(1) takes from baby's serum (N11) that 1 example never infected Epstein-Barr virus, do not detect VGA IgG; And in the serum sample that a routine infectious mononucleosis patients acuity infection period is got (N31), VCA IgG antibody has produced very early.Can be in order to do the quantitative of antibody objectively.
(2) (N31) no EBNA1 IgG antibody in infectious mononucleosis patients serum's sample in acute period.Just produce EBNA1 IgG (Chan KH etc.) generation VCA IgG antibody 8 months or more over a long time.
(3) therefore, occurring VCA IgG and EBNA1 IgG simultaneously is the index that infects Epstein-Barr virus in the past.
(4) compare with the healthy carrier, have the anti-Epstein-Barr virus antibody of wide spectrum among the nasopharyngeal carcinoma patients serum.
(5) antibody horizontal of EBNA1 IgG is higher than other antibody horizontal.This explanation, EBNA1 is an ebv infection, particularly nasopharyngeal carcinoma patient's a kind of important antigen.This also be with EBNA1 be that to colonize in the expressed major antigen of Epstein-Barr virus in the tumour cell corresponding to.
(6) in a word, The above results shows that enzyme linked immunosorbent assay can be used for differentiating the various Infection Status of Epstein-Barr virus.
(7) so, the enzyme linked immunosorbent assay of creating at the Epstein-Barr virus specific antigen when serodiagnosis Epstein-Barr virus relevant disease, has the very availability of potential advantages really.
4. optimize the ELISA method of serodiagnosis nasopharyngeal carcinoma
(1) point of interface of having created with sensitivity and specificity curve is that critical optical density value is owing to anti-Epstein-Barr virus antibody level in anti-Epstein-Barr virus antibody level among the nasopharyngeal carcinoma patients serum and the healthy carrier's serum has overlapped, can not have the absolute standard (seeing accompanying drawing 2) that to answer " sun " or " the moon ", the point of interface with sensitivity and specificity curve that we created is the method for critical optical density value (seeing accompanying drawing 3), distinguish " height " and " low ", just can reflect the feature of anti-Epstein-Barr virus antibody among the nasopharyngeal carcinoma patients serum objectively.
(2) created the dilutability (see figure 1) of determining to detect serum according to the titre curve of nasopharyngeal carcinoma patient group and health adult's group.
Table 3 is optimized the ELISA method of serodiagnosis nasopharyngeal carcinoma
VCA?IgG VCA?IgA?Zta-IgG Zta?IgA?EBNA1 EBNA1
IgG IgA
Anti-immunoglobulin, dilutability zG, 50k zA, 2k zG, 50k zA, 2k zG, 50k zA, 2k
Serum dilution * [n=81C, 74NRC] 100 200 20 2,000 100
Critical optical density value 1.25 0.83 11 0.62
Sensitivity 65% 86% 83% 72% 83%
Specificity 65% 86% 83% 72% 83%
*Data according to 74 routine nasopharyngeal carcinoma patients and 81 routine control groups get
Z: anti-immunoglobulin is available from Zymed, and zG represents Zymed IgG, and zA represents Zymed IgA
(3), in kit, provide a diagnosis reference serum that is equivalent to optimize critical optical density value for the judgement of diagnostic criteria.When this just uses the kit of same lot number to detect sample, can correct the relative optical density value of the high and low level of the applied differentiation of each batch detection in batches, draw more accurate and reliable data.
(4) owing to have the high-caliber anti-Epstein-Barr virus antibody of wide spectrum among the nasopharyngeal carcinoma patients serum mostly, the feature (see figure 4) of IgA antibody particularly, we have created two step detection methods, promptly at first use EBNA IgA, then carried out for second step again as belonging to " height " level, use Zta IgG with diagnosis, or make simultaneously with Zta-IgG and EBNA IgG with screening (seeing accompanying drawing 5).
In a word, the enzyme linked immunosorbent assay of new optimization (ELISA) has the specificity of objectivity and Epstein-Barr virus antigen, and it provides and has quantitatively picked up the hypotype of chaining pin to the different immune globulin antibodies of individual Epstein-Barr virus antigen.The enzyme linked immunosorbent assay of this optimization (ELISA) is new fully, is that traditional serological detection method is not accomplished, because the specificity that traditional serological detection method can not clear and definite antigen.And the enzyme linked immunosorbent assay of new optimization (ELISA) has adopted reference serum, can correct the difference between every batch of detection, has guaranteed the reliability of diagnosis.
5. the application of recombined EB virus antigen aspect diagnosis
5.1 the primary infection of Epstein-Barr virus
5.1.1 showing, immunofluorescence has VCA antibody; Because the EBNA antibody that postpones changes sun, so adopt the anticomplement immunofluorescence to show no EBNA antibody.
5.1.2 the EBV IgM positive.
5.1.3 low compatibility VCAIgG antibody
Here it is insensitive to be stressed that employing fluorescence immunoassay (IF) detects low compatibility VCA IgG, adopts ELISA then responsive; It is insensitive to adopt fluorescence immunoassay (IF) to detect VCA IgM, and adopts ELISA responsive.
In order to measure low compatibility VCA IgG antibody, a series of serum samples are worked on parallel emblem plate.A plate hole is handled 30min with 4M urea, and another plate hole is then not treated.When urea processing causing antibody horizontal descends more than 4 times or 4 times, show that then low compatibility VCA IgG antibody exists.
5.2 establish the antibody repertoire and the various ebv infections of antidiastole of ebv infection
The antibody repertoire of the various Infection Status of table 4 Epstein-Barr virus
1 2 3 4 5
VCA IgG EBNA IgG VCA IgM hangs down compatibility
Infection Status VCA IgG DNA example number
Primary infection
Positive negative positive 30
Positive negative positive negative 1
Positive negative positive 15
Subtotal 37 46
Recent infection
Positive negative negative 0 12
Subtotal 0 12
Past infects
Positive negative 14
Positive positive negative 1
Subtotal 1 15
Do not infect negative 08
Add up to 38 69
The antidiastole of the various ebv infection states of table 5
Enzyme linked immunosorbent assay
Immunofluorescence technique
The PCR method
Positive and the negative low compatibility p18 VCA IgG VCA IgM epstein barr virus dna of EBNAIgG of ebv infection state VCAIgG
Acute primary infection (n=46) 100% 97.8% 67.4% 80.4%
Recent infection (n=12) 100% 000
Past is infected (n=15) 0* 0 0.7% 0.7%
Do not infect (n=8) 0** 000
*All infected the people of Epstein-Barr virus in the past, detected VCA IgG and EBNA1 IgG through enzyme linked immunosorbent assay, all were positive.
*All did not infect the people of Epstein-Barr virus in the past, detected VCA IgG and EBNA1 IgG through enzyme linked immunosorbent assay, all were negative.
This shows,, adopt traditional immunofluorescence technique can not differentiate acute primary infection and recent infection because EBNA IgG will just change sun by a period of time serum antibody after infection; And adopting enzyme linked immunosorbent assay to detect low compatibility p18 VCA IgG of serum and VCAIgM, then most of acute primary infection patients (low compatibility p18 VCA IgG is 97.8%, and VCA IgM is 67.4%) can be positive.That is, adopt enzyme linked immunosorbent assay can differentiate acute primary infection and recent infection.
5.3 differentiate infectious mononucleosis (Infectious mononucleosis) and infectiousness monocyte syndrome (Infectious mononucleosis syndrome)
5.3.1 except that symptom, leucocyte raise (12000-18000), peripheral blood film is seen the CD4T lymphocyte that is not true to type, differently have a liking for anti-sheep red blood cell antibody (heteophil anti-sheepred blood cell antibodies), the smear EBERs in situ hybridization etc.
Refer to that 5.3.2 the acute table of infectious mononucleosis at first occurs promptly p18 VCA IgM and low compatibility VCA IgG are VCA IgG then, just occur EBNA1 IgG after 8 months.Therefore:
Do not infect Epstein-Barr virus: VCA feminine gender, EBNA1 are negative.
Infectious mononucleosis: the VCA positive, EBNA1 feminine gender.
Healthy carrier: the VCA positive, the EBNA1 positive.
VCA feminine gender, EBNA1 are negative, for not infecting Epstein-Barr virus.
Therefore, adopting the ELISA method detection EBNA1 IgG positive and the p18 VCA IgM positive then is infectious mononucleosis; Infectiousness monocyte syndrome then denys.
5.4 assisting diagnosis to doubt, serology is nasopharyngeal carcinoma
5.4.1 nasopharyngeal carcinoma patients serum's Epstein-Barr virus antibody horizontal feature and SD range of application
Table 6 nasopharyngeal carcinoma patients serum Epstein-Barr virus antibody horizontal feature and SD range of application
The SD range of application of nasopharyngeal carcinoma patients serum's Epstein-Barr virus antibody horizontal feature
The serum Epstein-Barr virus of wide spectrum is anti-stops, particularly the rising 1. serodiagnosis nasopharyngeal carcinoma of IgA
2. mass survey is with the early detection nasopharyngeal carcinoma
The curative effect of epstein barr virus dna 1. predicted treatments in the blood serum of high copy amount
5.4.2 the independent use of immunofluorescence technique and enzyme linked immunosorbent assay
(the lymphoepithelioma sample cancer beyond 51 routine nasopharyngeal carcinoma, the 4 routine nasopharynxs, 23 other cancers of example and 140 routine other diseases) have done following analysis in the serum sample of Hong Kong Mary hospital 218 routine continuous detecting.
The comparison that table 7 immunofluorescence technique and enzyme linked immunosorbent assay are used separately
The negative prediction of the old property of detection method nasopharyngeal carcinoma control group prediction practicality
True positives/false negative false positive/true negative
Immunofluorescence:
VCA IgA 5,1/4 65,/98 44% 96% gets rid of and (prediction)
EA IgA 40,/15 5/,158 89% 91% predictions
Enzyme linked immunological absorption:
EBNA1 IgA 4,6/9 22/,141 68% 94% (eliminating) and predictions
Zta?IgG 41/14 28/135 59％ 91％
EBV DNA 31,/24 3/,160 91% 87% predictions
Positive prediction=true positives/true positives+false positive * 100%=A/ (A+C) * 100%
Negative prediction=true negative/false negative+true negative * 100%=D/ (B+D) * 100%
5.4.3 the comparison of uniting use of immunofluorescence technique and enzyme linked immunosorbent assay
(the lymphoepithelioma sample cancer beyond 51 routine nasopharyngeal carcinoma, the 4 routine nasopharynxs, 23 other cancers of example and 140 routine other diseases) have done following analysis equally in the serum sample of Hong Kong Mary hospital 218 routine continuous detecting.
Table 8 immunofluorescence technique is united the comparison of use aspect the serodiagnosis nasopharyngeal carcinoma
The negative prediction of the old property of nasopharyngeal carcinoma group health adult control group prediction
True positives false negative false positive true negative
The VCAIgA positive+EAIgA positive 40 15 5 158 88.9%
The VCAIgA positive+EAIgA negative 11 44 64 99 14.7%
VCAIgA feminine gender+EAIgA positive 0 55 0 163
VCAIgA feminine gender+EAIgA negative 51 4 89 94 95.9%
Positive prediction=true positives/(true positives+false positive) * 100%=TP/ (TP+FP)
40/(40+5)＝88.9％
11/(11+64)＝14.7％
Negative prediction=true negative/(false negative+true negative) * 100%=TN/ (TN+FN)
94/(94+4)＝95.9％
It is worthy of note, no matter be nasopharyngeal carcinoma patient or health adult, do not have an example to be VCA IgA feminine gender among the EA IgA positive person, this is because the antigen in existing dissolving late period among " VCA " that the B95-8 cell that adopted is produced has comprised again and dissolved early stage antigen.So the antigentic specificity of the VCA IgA that is adopted in immunofluorescence technique and the antigentic specificity of EA IgA are overlapped.That is to say, used two kinds of immunofluorescence techniques though unite, can not reach the effect of " 1+1=2 ", promptly complementation is not strong.And in VCA IgA and the EAIgA jack to jack adapter 98,4 examples are false negative, and false negative rate reaches 4.1% (4/4+94), can not play the effect of effective eliminating diagnosis nasopharyngeal carcinoma clinically.The uniting use and but can overcome this shortcoming of the following stated enzyme linked immunosorbent assay.
5.4.4 be the EBNA1 IgA of enzyme linked immunosorbent assay earlier, be immunofluorescence VCA IgA again
Table 9
The EBNA1 IgA of the immunofluorescence VCA IgA enzyme linked immunosorbent assay of enzyme linked immunosorbent assay
EBNA1 IgA and immunofluorescence VCA IgA associating
Sensitivity 89% 86% 88%
Specificity 78% 91% 95%
Table 10 enzyme linked immunosorbent assay is united the comparison of use
The negative prediction of the old property of nasopharyngeal carcinoma group health adult control group prediction
True positives false negative false positive true negative
The EBNA1 IgA positive+ZtaIgG positive 33 22 5 158 86.8%
The EBNA1 IgA positive+ZtaIgG negative 13 42 17 146 43.3%
EBNA1 IgA feminine gender+ZtaIgG positive 8 47 25 138 24.2%
EBNA1 IgA feminine gender+ZtaIgG negative 54 1 47 116 99.1%
Positive prediction=true positives/(true positives+false positive) * 100%=TP/ (TP+FP)
33/(33+5)＝88.8％
13/(13+17)＝43.3％
8/(8+25)＝
Negative prediction=true negative/(false negative+true negative) * 100%=TN/ (TN+FN)
116/(1+116)＝
Relatively as can be known, the negative predictive value (99.1%) that enzyme linked immunosorbent assay is united use (EBNA1 IgA+Zta IgG) is much higher than immunofluorescence technique unites use (VCA IgA+EAIgA), promptly unite clinically when using EBNA IgA and Zta IgG, if jack to jack adapter, the reliability of then getting rid of nasopharyngeal carcinoma diagnosis reaches 99.1%.Two kinds of enzyme linked immunosorbent assay joint-detection have very strong complementation when the serodiagnosis nasopharyngeal carcinoma.
5.4.5 adopt the association between immunofluorescence and the employing enzyme linked immunological absorption serodiagnosis nasopharyngeal carcinoma
Table 11 adopts the association between immunofluorescence and the employing enzyme linked immunological absorption serodiagnosis nasopharyngeal carcinoma
Immunofluorescence serodiagnosis enzyme linked immunological absorption serodiagnosis percent
Mutually identical 100 examples 64.5%
51 examples 32.9% equal to each other
Misfit 4 examples 2.6% mutually
Amount to 155 examples
5.4.6 the prediction and the eliminating of the serodiagnosis nasopharyngeal carcinoma of enzyme linked immunological absorption use in conjunction
Table 12
Example number EBNA1 Zta IgG prediction comment
IgA
33 is high positive, and 87% meets nasopharyngeal carcinoma consistently
30 height are positive, meet to 43% moderate nasopharyngeal carcinoma
33 is low high positive, and 24% meets nasopharyngeal carcinoma critically
117 is low negative, and 99% does not meet nasopharyngeal carcinoma consistently
5.5 the risk of nasopharyngeal carcinoma is suffered from early detection and assessment
Use in conjunction EBNA1 IgA, EBNA1 IgG and Zta IgG show that odds ratio increases greatly.
Table 13 use in conjunction EBNA1 IgA, EBNA1 IgG and Zta IgG show that odds ratio increases greatly
The observed % of Epstein-Barr virus antibody repertoire (% of expectation)
EBNA1 IgA EBNA1 IgG Zta IgG nasopharyngeal carcinoma (n=121) health adult (n=332) odds ratio
Low low 0.83 (0.54) 59.3 (59.17) 0.009
Low high by 5.79 (2.01) 14.8 (14.79) 0.13
Low height 1.65 (2.61) 8.73 (9.63) 0.28
Height low 3.31 (3.03) 10.2 (9.63) 0.31
Low high by 6.61 (9.84) 3.01 (2.41) 3.98
Height is high by 6.61 (11.42) 1.51 (2.41) 4.50
Height 15.7 (14.82) 1.51 (1.57) 9.54
Gao Gaogao 59.5 (55.73) 0.9 (0.39) 137.9
Sum 100 100 1.0
r＝0.990 r＝0.999
The examination of two levels:
First level: EBNAIgA
Second level: Zta IgG+EBNA IgG
The examination of two levels of the risk of table 14 early detection and assessment trouble nasopharyngeal carcinoma
The horizontal relative risk degree of/1 000 EBNA1 IgA EBNA1 IgG Zta IgG risk is followed up a case by regular visits to
4 Gao Gaogaogao 138 doubt and are nasopharyngeal carcinoma
9 risks that should guard in 16 height
The risk that 24 height senior middle schools 4 should guard
The low 0.3 little risk of 96 height
860 low do not do low 0.2 and forget it
140 people (14.0%, 140/1000) think to have the risk of suffering from nasopharyngeal carcinoma in 1000 people.In this 140 people: 4 people (2.86%, 4/140) think to have the excessive risk degree; 40 people (28.57%, 40/140) think to have the medium risk degree; 96 people (68.57%, 96/140) think to have little risk.860 people EBNA1 IgA are low, further are not Zta-IgG and EBNA1 IgG, think the devoid of risk degree.
In addition the examination of Hong Kong private organization Hong Kong resident 2000 people, find that 6 examples have the people of excessive risk degree, wherein 1 people turns out to be the loyal patient of nasopharyngeal carcinoma, does not have recurrence after treating in 5 years; The every other nasopharyngeal carcinoma of not suffering from per capita.
5.6 assist diagnosis Burkitt lymthoma
Use Zta IgGl.
EBV-1.ST25
Epstein-Barr virus enzyme linked immunological absorption diagnostic kit and preparation method thereof
Sequence table (SEQUENCE LISTING)
＜110〉Hongkong Shennong Co., Ltd. (Sinoclone Limited)
＜120〉Epstein-Barr virus enzyme linked immunological absorption diagnostic kit and preparation method thereof
<130>EBV?Serological?Diagnositic?Kits
<160>6
<170>PatentIn?version?3.1
<210>1
<211>705
<212>DNA
<213>Epstein-Barr?Virus
<400>1
cctgtagggg?aagccgatta?ttttgaatac?caccaagaag?gtggcccaga?tggtgagcct 60
gacgtgcccc?cgggagcgat?agagcagggc?cccgcagatg?acccaggaga?aggcccaagc 120
actggacccc?ggggtcaggg?tgatggaggc?aggcgcaaaa?aaggagggtg?gtttggaaag 180
catcgtggtc?aaggaggttc?caacccgaaa?tttgagaaca?ttgcagaagg?tttaagagct 240
ctcctggcta?ggagtcacgt?agaaaggact?accgacgaag?gaacttgggt?cgccggtgtg 300
ttcgtatatg?gaggtagtaa?gacctccctt?tacaacctaa?ggcgaggaac?tgcccttgct 360
attccacaat?gtcgtcttac?accattgagt?cgtctcccct?ttggaatggc?ccctggaccc 420
ggcccacaac?ctggcccgct?aagggagtcc?attgtctgtt?atttcatggt?ctttttacaa 480
actcatatat?ttgctgaggt?tttgaaggat?gcgattaagg?accttgttat?gacaaagccc 540
gctcctacct?gcaatatcag?ggtgactgtg?tgcagctttg?acgatggagt?agatttgcct 600
ccctggtttc?cacctatggt?ggaaggggct?gccgcggagg?gtgatgacgg?agatgacgga 660
gatgaaggag?gtgatggaga?tgagggtgag?gaagggcagg?agtga 705
<210>2
<211>234
<212>PRT
<213>Epstein-Barr?Virus
<400>2
Pro?Val?Gly?Glu?Ala?Asp?Tyr?Phe?Glu?Tyr?His?Gln?Glu?Gly?Gly?Pro
1 5 10 15
Asp?Gly?Glu?Pro?Asp?Val?Pro?Pro?Gly?Ala?Ile?Glu?Gln?Gly?Pro?Ala
20 25 30
Asp?Asp?Pro?Gly?Glu?Gly?Pro?Ser?Thr?Gly?Pro?Arg?Gly?Gln?Gly?Asp
35 40 45
Gly?Gly?Arg?Arg?Lys?Lys?Gly?Gly?Trp?Phe?Gly?Lys?His?Arg?Gly?Gln
50 55 60
Gly?Gly?Ser?Asn?Pro?Lys?Phe?Glu?Asn?Ile?Ala?Glu?Gly?Leu?Arg?Ala
65 70 75 80
Leu?Leu?Ala?Arg?Ser?His?Val?Glu?Arg?Thr?Thr?Asp?Glu?Gly?Thr?Trp
85 90 95
EBV-1.ST25
Val?Ala?Gly?Val?Phe?Val?Tyr?Gly?Gly?Ser?Lys?Thr?Ser?Leu?Tyr?Asn
100 105 110
Leu?Arg?Arg?Gly?Thr?Ala?Leu?Ala?Ile?Pro?Gln?Cys?Arg?Leu?Thr?Pro
115 120 125
Leu?Ser?Arg?Leu?Pro?Phe?Gly?Met?Ala?Pro?Gly?Pro?Gly?Pro?Gln?Pro
130 135 140
Gly?Pro?Leu?Arg?Glu?Ser?Ile?Val?Cys?Tyr?Phe?Met?Val?Phe?Leu?Gln
145 150 155 160
Thr?His?Ile?Phe?Ala?Glu?Val?Leu?Lys?Asp?Ala?Ile?Lys?Asp?Leu?Val
165 170 175
Met?Thr?Lys?Pro?Ala?Pro?Thr?Cys?Asn?Ile?Arg?Val?Thr?Val?Cys?Ser
180 185 190
Phe?Asp?Asp?Gly?Val?Asp?Leu?Pro?Pro?Trp?Phe?Pro?Pro?Met?Val?Glu
195 200 205
Gly?Ala?Ala?Ala?Glu?Gly?Asp?Asp?Gly?Asp?Asp?Gly?Asp?Glu?Gly?Gly
210 215 220
Asp?Gly?Asp?Glu?Gly?Glu?Glu?Gly?Gln?Glu
225 230
<210>3
<211>531
<212>DNA
<213>Epstein-Barr?Virus
<400>3
atggcacgcc?ggctgcccaa?gcccaccctc?caggggaggc?tggaggcgga?ttttccagac 60
agtcccctgc?ttcctaaatt?tcaagagctg?aaccagaata?atctccccaa?tgatgttttt 120
cgggaggctc?aaagaagtta?cctggtattt?ctgacatccc?agttctgcta?cgaagagtac 180
gtgcagagga?cttttggggt?gcctcggcgc?caacgcgcca?tagacaagag?gcagagagcc 240
agtgtggctg?gggctggtgc?tcatgcacac?cttggcgggt?catccgccac?ccccgtccag 300
caggctcagg?ccgccgcatc?cgctgggacc?ggggccttgg?catcatcagc?gccgtccacg 360
gccgtagccc?agtccgcgac?cccctctgtt?tcttcatcta?ttagcagcct?ccgggccgcg 420
acttcggggg?cgactgccgc?cgcctccgcc?gccgcagccg?tcgataccgg?gtcaggtggc 480
gggggacaac?cccacgacac?cgccccacgc?ggggcacgta?agaaacagta?g 531
<210>4
<211>176
<212>PRT
<213>Epstein-Barr?Virus
<400>4
Met?Ala?Arg?Arg?Leu?Pro?Lys?Pro?Thr?Leu?Gln?Gly?Arg?Leu?Glu?Ala
1 5 10 15
Asp?Phe?Pro?Asp?Ser?Pro?Leu?Leu?Pro?Lys?Phe?Gln?Glu?Leu?Asn?Gln
20 25 30
EBV-1.ST25
Asn?Asn?Leu?Pro?Asn?Asp?Val?Phe?Arg?Glu?Ala?Gln?Arg?Ser?Tyr?Leu
35 40 45
Val?Phe?Leu?Thr?Ser?Gln?Phe?Cys?Tyr?Glu?Glu?Tyr?Val?Gln?Arg?Thr
50 55 60
Phe?Gly?Val?Pro?Arg?Arg?Gln?Arg?Ala?Ile?Asp?Lys?Arg?Gln?Arg?Ala
65 70 75 80
Ser?Val?Ala?Gly?Ala?Gly?Ala?His?Ala?His?Leu?Gly?Gly?Ser?Ser?Ala
85 90 95
Thr?Pro?Val?Gln?Gln?Ala?Gln?Ala?Ala?Ala?Ser?Ala?Gly?Thr?Gly?Ala
100 105 110
Leu?Ala?Ser?Ser?Ala?Pro?Ser?Thr?Ala?Val?Ala?Gln?Ser?Ala?Thr?Pro
115 120 125
Ser?Val?Ser?Ser?Ser?Ile?Ser?Ser?Leu?Arg?Ala?Ala?Thr?Ser?Gly?Ala
130 135 140
Thr?Ala?Ala?Ala?Ser?Ala?Ala?Ala?Ala?Val?Asp?Thr?Gly?Ser?Gly?Gly
145 150 155 160
Gly?Gly?Gln?Pro?His?Asp?Thr?Ala?Pro?Arg?Gly?Ala?Arg?Lys?Lys?Gln
165 170 175
<210>5
<211>738
<212>DNA
<213>Epstein-Barr?Virus
<400>5
atgatggacc?caaactcgac?ttctgaagat?gtaaaattta?cacctgaccc?ataccaggtg 60
ccttttgtac?aagcttttga?ccaagctacc?agagtctatc?aggacctggg?agggccatcg 120
caagctcctt?tgccttgtgt?gctgtggccg?gtgctgccag?agcctctgcc?acaaggccag 180
ctaactgcct?atcatgtttc?aaccgctccg?actgggtcgt?ggttttctgc?ccctcagcct 240
gctcctgaga?atgcttatca?agcttatgca?gcacctcagc?tgttcccagt?ctccgacata 300
acccagaatc?aacagactaa?ccaagccggg?ggagaagcac?ctcaacctgg?agacaattct 360
actgttcaaa?cagcagcagc?agtggtgttt?gcttgccccg?gggctaacca?aggacaacag 420
ctagcagaca?ttggtgttcc?acagcctgca?ccagtggctg?ccccggcacg?acgcacacgg 480
aaaccacaac?agccagaatc?gttggaggaa?tgcgattctg?aactagaaat?aaagcgatac 540
aagaatcggg?tggcttccag?aaaatgccgg?gccaagttta?agcaactgct?gcagcactac 600
cgtgaggtgg?ctgctgccaa?atcatctgaa?aatgacaggc?tgcgcctcct?gttgaagcag 660
atgtgcccaa?gcctggatgt?tgactccatt?atcccccgga?caccagatgt?tttacacgag 720
gatctcttaa?atttctaa 738
<210>6
<211>245
<212>PRT
<213>Epstein-Barr?Virus
EBV-1.ST25
<400>6
Met?Met?Asp?Pro?Asn?Ser?Thr?Ser?Glu?Asp?Val?Lys?Phe?Thr?Pro?Asp
1 5 10 15
Pro?Tyr?Gln?Val?Pro?Phe?Val?Gln?Ala?Phe?Asp?Gln?Ala?Thr?Arg?Val
20 25 30
Tyr?Gln?Asp?Leu?Gly?Gly?Pro?Ser?Gln?Ala?Pro?Leu?Pro?Cys?Val?Leu
35 40 45
Trp?Pro?Val?Leu?Pro?Glu?Pro?Leu?Pro?Gln?Gly?Gln?Leu?Thr?Ala?Tyr
50 55 60
His?Val?Ser?Thr?Ala?Pro?Thr?Gly?Ser?Trp?Phe?Ser?Ala?Pro?Gln?Pro
65 70 75 80
Ala?Pro?Glu?Ash?Ala?Tyr?Gln?Ala?Tyr?Ala?Ala?Pro?Gln?Leu?Phe?Pro
85 90 95
Val?Ser?Asp?Ile?Thr?Gln?Asn?Gln?Gln?Thr?Asn?Gln?Ala?Gly?Gly?Glu
100 105 110
Ala?Pro?Gln?Pro?Gly?Asp?Asn?Ser?Thr?Val?Gln?Thr?Ala?Ala?Ala?Val
115 120 125
Val?Phe?Ala?Cys?Pro?Gly?Ala?Asn?Gln?Gly?Gln?Gln?Leu?Ala?Asp?Ile
130 135 140
Gly?Val?Pro?Gln?Pro?Ala?Pro?Val?Ala?Ala?Pro?Ala?Arg?Arg?Thr?Arg
145 150 155 160
Lys?Pro?Gln?Gln?Pro?Glu?Ser?Leu?Glu?Glu?Cys?Asp?Ser?Glu?Leu?Glu
165 170 175
Ile?Lys?Arg?Tyr?Lys?Asn?Arg?Val?Ala?Ser?Arg?Lys?Cys?Arg?Ala?Lys
180 185 190
Phe?Lys?Gln?Leu?Leu?Gln?His?Tyr?Arg?Glu?Val?Ala?Ala?Ala?Lys?Ser
195 200 205
Ser?Glu?Asn?Asp?Arg?Leu?Arg?Leu?Leu?Leu?Lys?Gln?Met?Cys?Pro?Ser
210 215 220
Leu?Asp?Val?Asp?Ser?Ile?Ile?Pro?Arg?Thr?Pro?Asp?Val?Leu?His?Glu
225 230 235 240
Asp?Leu?Leu?Asn?Phe
245

Claims (16)

1, a kind of Epstein-Barr virus enzyme linked immunological absorption diagnostic kit, include: positive control serum, negative control sera, zymolyte, cessation reaction liquid, dilution buffer liquid, dcq buffer liquid and Epstein-Barr virus target antigen is characterized in that the Epstein-Barr virus antigen protein is: EBNA1 (BKRF1) albumen, Zta (BZLF1) albumen and VCA-p18 albumen (being VCA or the BFRF3 albumen of 18Kd).
2,, it is characterized in that selected EBNA1 amino acid sequence and following sequence have 95% homology according to the described kit of claim 1:
MSDEGPGTGPGNGLGEKGDTSGPEGSGGSGPQRRGGDNHGRGRG
RGRGRGGGRPGAPGGSGSGPRHRDGVRRPQKRPSCIGCKGTHGGTGAGAGAGGAGAGG
AGAGGGAGAGGGAGGAGGAGGAGAGGGAGAGGGAGGAGGAGAGGGAGAGGGAGGAGAG
GGAGGAGGAGAGGGAGAGGGAGGAGAGGGAGGAGGAGAGGGAGAGGAGGAGGAGAGGA
GAGGGAGGAGGAGAGGAGAGGAGAGGAGAGGAGGAGAGGAGGAGAGGAGGAGAGGGAG
GAGAGGGAGGAGAGGAGGAGAGGAGGAGAGGAGGAGAGGGAGAGGAGAGGGGRGRGGS
GGRGRGGSGGRGRGGSGGRRGRGRERARGGSRERARGRGRGRGEKRPRSPSSQSSSSG
SPPRRPPPGRRPFFH PVGEADYFEYHQEGGPDGEPDVPPGAIEQGPADDPGEGPSTGP
RGQGDGGRRKKGGWFGKHRGQGGSNPKFENIAEGLRALLARSHVERTTDEGTWVAGVF
VYGGSKTSLYNLRRGTALAIPQCRLTPLSRLPFGMAPGPGPQPGPLRESTVCYFMVFL
QTHIFAEVLKDAIKDLVMTKPAPTCNIRVTVCSFDDGVDLPPWFPPMVEGAAAEGDDG
DDGDEGGDGDEGEEGQE
Wherein tool is distinctive to be back one section, that is:
PVGEADYFEYHQEGGPDGEPDVPPGAIEQGPADDPGEGPSTGP
RGQGDGGRRKKGGWFGKHRGQGGSNPKFENIAEGLRALLARSHVERTTDEGTWVAGVF
VYGGSKTSLYNLRRGTALAIPQCRLTPLSRLPFGMAPGPGPQPGPLRESIVCYFMVFL
QTHIFAEVLKDAIKDLVMTKPAPTCNIRVTVCSFDDGVDLPPWFPPMVEGAAAEGDDG
DDGDEGGDGDEGEEGQE
3,, it is characterized in that selected Zta amino acid sequence and following sequence have 95% homology according to the described kit of claim 1:
MMDPNSTSEDVKFTPDPYQVPFVQAFDQATRVYQDLGGPSQAPL
PCVLWPVLPEPLPQGQLTAYHVSTAPTGSWFSAPQPAPENAYQAYAAPQLFPVSDITQ
NQQTNQAGGEAPQPGDNSTVQTAAAVVFACPGANQGQQLADIGVPQPAPVAAPARRTR
KPQQPESLEECDSELEIKRYKNRVASRKCRAKFKQLLQHYREVAAAKSSENDRLRLLL
KQMCPSLDVDSIIPRTPDVLHEDLLNF
4,, it is characterized in that selected VCA-p18 amino acid sequence and following sequence have 95% homology according to the described kit of claim 1:
MARRLPKPTLQGRLEADFPDSPLLPKFQELNQNNLPNDVFREAQ
RSYLVFLTSQFCYEEYVQRTFGVPRRQRAIDKRQRASVAGAGAHAHLGGSSATPVQQA
QAAASAGTGALASSAPSTAVAQSATPSVSSSISSLRAATSGATAAASAAAAVDTGSGG
GGQPHDTAPRGARKKQ
5,, it is characterized in that it also includes the diagnosis reference serum that is equivalent to optimize critical optical density value according to the described kit of claim 1.
6,, it is characterized in that reference serum is best critical value with the relative optical density value (rOD) of sensitivity curve and specificity curve interface point according to the described kit of claim 5.
7, the preparation method of Epstein-Barr virus enzyme linked immunological absorption diagnostic kit, preparation, purifying mark, the titration of tiring, the bag that comprises recombinant antigen be is characterized in that by plate preparation, sealing, drying and packing, the preparation of enzyme labelled antibody concentrate, dilution preparation, positive control preparation, normal control preparation, substrate packing, stop buffer and concentrate step such as purchase it also comprises the preparation of reference serum.
8,, it is characterized in that preparing reference serum for best critical value with the relative optical density value (rOD) of sensitivity curve and specificity curve interface point according to the preparation method of the described kit of claim 7.
9,, it is characterized in that being prepared as of Epstein-Barr virus albumen recombinant antigen according to the preparation method of the described kit of claim 7:
A, selected EBNA1 (BKRF1) albumen back one section, that is:
PVGEADYFEYHQEGGPDGEPDVPPGAIEQGPADDPGEGPSTGP
RGQGDGGRRKKGGWFGKHRGQGGSNPKFENIAEGLRALLARSHVERTTDEGTWVAGVF
VYGGSKTSLYNLRRGTALAIPQCRLTPLSRLPFGMAPGPGPQPGPLRESIVCYFMVFL
QTHIFAEVLKDAIKDLVMTKPAPTCNIRVTVCSFDDGVDLPPWFPPMVEGAAAEGDDG
DDGDEGGDGDEGEEGQE
And Zta (BZLF1) albumen and the mRNA sequence of VCA-p18 albumen (being VCA or the BFRF3 albumen of 18Kd) these three kinds of Epstein-Barr virus albumen in gene pool (V01555, the sequence of B95-8 cell line), reverse transcriptase to the cDNA sequence of transferring;
B, design can increase primer, the sequence amplification of this cDNA sequence;
C, with pcr amplification and glutathione transferase (GST) gene clone to the procaryotic cell expression pGEX2T plasmid vector of Eco R1/Bam HI digestion, the plasmid of reorganization is gone to Escherichia coli, induce the Escherichia coli that forwarded recombinant plasmid to, gather in the crops then and the cracking bacterium, the bacterium pyrolysis product is removed fragment after centrifugal and is clarified, and the supernatant that contains fusion adopts glutathione agarose 4B through the paddy Guang
Sweet peptidyl transferase-glutathione affinity chromatography instrument purifying.
10,, it is characterized in that EBNA-1 (BKRF1) design of primers is according to the preparation method of the described kit of claim 7:
5’-TAC? GGA?TCC?CCT?GTA?GGG?GAA?GCC?GAT?TA-3’
BamHI
5’-TAC? GAA?TTC?TCA?CTC?CTG?CCC?TTC?CTC?CAC-3’
EcoRI
11,, it is characterized in that Zta (BZLF1) design of primers is according to the preparation method of the described kit of claim 7:
5’-TAC? GGA?TCC?ATG?ATG?GAC?CCA?AAC?TCG?AC-3’
BamHI
5’-TAC? GAA?TTC?TTA?GAA?ATT?TAA?GAG?ATC?CTC-3’
EcoRI
12,, it is characterized in that VCA-p18 (BFRF3) design of primers is according to the preparation method of the described kit of claim 7:
5’-TAC? GGA?TCC?ATG?GCA?CGC?CGG?CTG?CCC?AAG-3’
BamHI
5’-TAC? GAA?TTC?CTA?CTG?TTT?CTT?ACG?TG-3’
EcoRI
13, EBNA1 (BKRF1) albumen, Zta (BZLF1) albumen separately or use in conjunction in the serodiagnosis of nasopharyngeal carcinoma.
14, EBNA1 (BKRF1) albumen, Zta (BZLF1) albumen and VCA-p18 albumen use in conjunction are in the serodiagnosis of nasopharyngeal carcinoma.
15, the application of VCA-p18 albumen in the serodiagnosis of infectious mononucleosis.
16, EBNA1 (BKRF1) albumen and the use in conjunction of VCA-p18 albumen in the serodiagnosis of Epstein-Barr virus recent infection.
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