CN1793929A - Competitive enzyme-linked immune detection method of hepatitis E virus neutralization antibody - Google Patents
Competitive enzyme-linked immune detection method of hepatitis E virus neutralization antibody Download PDFInfo
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Abstract
A competition enzyme - linked immune detection method of neutralization antibody on hepatitis virus in the fifth type includes competing and combining neutralization antibody resisting hepatitis virus in the fifth type with neutralization single clone antibody of enzyme labeled hepatitis virus in the fifth type to encapsulate neutralization antigen tabular position on antigen, realizing competition enzyme - linked immune detection of neutralization antibody on the fifth type of hepatitis virus in biological sample according to color developing decrease degree of enzyme action substrate.
Description
Technical field
The present invention relates to belong to hepatitis E virus neutralizing antibody detection method in the serum (slurry) of ELISA detection method of serum (slurry) antibody, relate in particular to the competitive enzyme-linked immune detection method of hepatitis E virus neutralizing antibody.
Technical background
(hepatitis E virus is nonencapsulated single positive chain RNA virus HEV) to hepatitis E virus, propagates through fecal-oral route, acute Hepatitis E (the hepatitis E that causes, HE), show as acute icteric hepatitis more, easily develop into subacute hepatitis sequestrans or silt courage hepatitis.According to Ministry of Health of the People's Republic of China's 27 kinds of statutory report epidemic reports statistics, HE incidence of disease height, the mortality ratio height is ascendant trend year by year in recent years.The average case fatality rate of HE in November, 2005 was about 0.37% in 2002～, far beyond the hepatitis A of propagating through fecal-oral route equally (0.06%) is high, height ranks first in virus hepatitis, especially the pregnant woman is ill, case fatality rate is up to 20% even 35%, and Susceptible population mostly is 15～59 years old person between twenty and fifty, and harm is serious.HE does not still have special methods of treatment at present, and also not having active and passive immunity preparation can be for prevention.Chinese scholars is devoted to the research of hepatitis E virus vaccine.The validity of vaccine is to induce body to produce neutralizing antibody; with on the virus structural protein in and epitope combine; block the corresponding receptors bind on the cell membrane of this epi-position and sensitive cells, thereby stop this poisoning intrusion sensitive cells, the protective effect of performance vaccine.And weigh whether effectively standard of vaccine, be exactly that can vaccine induce the generation neutralizing antibody, and induce the ability that produces neutralizing antibody, tradition adopt in the body and (or) external neutralization test identifies the validity of vaccine.Animal to the hepatitis E virus sensitivity has only non-human primate at present, yet non-human primate mostly is subclinical type infection after infecting hepatitis E virus, be difficult to obtain satisfied animal model, and use non-human primate to set up animal model, and costing an arm and a leg, method is loaded down with trivial details, test period is long, animal feeding requires high, expends lot of manpower and material resources, is difficult to be extensive use of in basic unit.In addition, because hepatitis E virus lacks effective tissue culturing system, traditional external neutralization test is identified and is difficult to carry out, Meng Ji letters in 1998 etc. have been invented the external neutralization test of PCR-based and cellular incubation in order to estimate the validity of hepatitis E virus vaccine, but this external neutralization test needs cellular incubation and RT-PCR technology, method is loaded down with trivial details, and price is also expensive, also is difficult to generally apply.Thereby develop a kind of method of simple and easy to do detection hepatitis E virus neutralizing antibody, be to be the task of top priority.
Summary of the invention
The objective of the invention is: develop hepatitis E virus neutralizing antibody competitive enzyme-linked immune detection method, replace in the hepatitis E virus body and (or) external neutralization test, overcome in the body and (or) the neutralization test method is loaded down with trivial details, expensive, time-consuming, the shortcoming of effort, the validity of evaluation vaccine.
The present invention is achieved in that a kind of competitive enzyme-linked immune detection method of hepatitis E virus neutralizing antibody: serum (slurry) or other biological imitate anti-hepatitis E virus neutralizing antibody and enzyme labeling hepatitis E virus neutralizing monoclonal antibody in this compete combine on the envelope antigen in and epitope, according to the degree that the zymolyte chromogenic reaction weakens, realize the competitive enzyme-linked immune of the hepatitis E virus neutralizing antibody in the biological sample is detected.
Above-mentioned envelope antigen is the hepatitis E virus open reading frame 2 of different genotype and hypotype or the recombinant protein or the polypeptide of its fragment coding, can form envelope antigen alone or in combination.
Good effect of the present invention: one, easy to be quick: as to use competition law ELISA and detect the hepatitis E virus neutralizing antibody, simple and easy to do, can carry out whenever and wherever possible, overcome in the body and (or) external neutralization test method is loaded down with trivial details, the shortcoming that wastes time and energy, can be used for the experimental evaluation of scientific research institutions' exploitation vaccine, also can be applicable in basic unit.Two, high specificity: owing to use the hepatitis E virus neutralizing monoclonal antibody as the competition bond, that can compete with it only has a hepatitis E virus neutralizing antibody, thereby has the specificity of height.Three, cost is low, security good: owing to use the ELISA method; it is low to use reagent production operation cost in the experiment; overcome neutralization test,in vivo and used non-human primate; cost an arm and a leg, consuming time, the consumption power shortcoming; and virion or viral nucleic acid that must the tactility metachromia; can not pollute any environment, security is good.Four, social benefit is good: hepatitis e virus infection is ascendant trend year by year, HE case fatality rate height, between twenty and fifty susceptible, harm is serious, the application of vaccine is the important measures that keep off infection, and sets up simple and effective vaccine evaluation method, can estimate preferably the validity of vaccine, reduce society's control cost, have good social benefit.
Description of drawings
Fig. 1 be competitive ELISA that the embodiment of the invention adopts detect hepatitis E virus vaccine immunity rhesus macaque serum anti--figure as a result of hepatitis E virus neutralizing antibody.
Fig. 2 is a hepatitis E virus neutralizing antibody competitive enzyme-linked immune detection method schematic flow sheet of the present invention.
Embodiment
Following examples describe the present invention in detail:
1, the acquisition of envelope antigen
Envelope antigen can form envelope antigen alone or in combination from the hepatitis E virus open reading frame 2 of different genotype and hypotype or the recombinant protein or the polypeptide of its fragment coding.Be the acquisition that example describes envelope antigen in detail with 7 representative strain hepatitis E virus below.
7 strain hepatitis E virus are respectively the hepatitis E virus Pakistan strain (M80581) from the hepatitis E virus Burma strain (M73218) of I genotype Ia hypotype, Ib hypotype, the hepatitis E virus Morocco strain (AY230202) of Ic hypotype, from the genotypic hepatitis E virus of II Mexico's strain (M74506), from the genotypic hepatitis E virus of III U.S.'s strain (AF060668) and hepatitis E virus New Zealand pig strain (AF082843), and from the genotypic hepatitis E virus Chinese strain of IV (AJ272108).
7 strain hepatitis E virus ORF2 coding nucleotide sequences are respectively:
Hepatitis E virus Burma strain (M 73218) ORF2:
Atgcgccctcggcctattttgttgctgctcctcatgtttttgcctatgctgcccgcgccaccgcccggtcagccgtctggccgccgtcgtgggcggcgcagcggcggttccggcggtggtttctggggtgaccgggttgattctcagcccttcgcaatcccctatattcatccaaccaaccccttcgcccccgatgtcaccgctgcggccggggctggacctcgtgttcgccaacccgcccgaccactcggctccgcttggcgtgaccaggcccagcgccccgccgttgcctcacgtcgtagacctaccacagctggggccgcgccgctaaccgcggtcgctccggcccatgacaccccgccagtgcctgatgtcgactcccgcggcgccatcttgcgccggcagtataacctatcaacatctccccttacctcttccgtggccaccggcactaacctggttctttatgccgcccctcttagtccgcttttaccccttcaggacggcaccaatacccatataatggccacggaagcttctaattatgcccagtaccgggttgcccgtgccacaatccgttaccgcccgctggtccccaatgctgtcggcggttacgccatctccatctcattctggccacagaccaccaccaccccgacgtccgttgatatgaattcaataacctcgacggatgttcgtattttagtccagcccggcatagcctctgagcttgtgatcccaagtgagcgcctacactatcgtaaccaaggctggcgctccgtcgagacctctggggtggctgaggaggaggctacctctggtcttgttatgctttgcatacatggctcactcgtaaattcctatactaatacaccctataccggtgccctcgggctgttggactttgcccttgagcttgagtttcgcaaccttacccccggtaacaccaatacgcgggtctcccgttattccagcactgctcgccaccgccttcgtcgcggtgcggacgggactgccgagctcaccaccacggctgctacccgctttatgaaggacctctattttactagtactaatggtgtcggtgagatcggccgcgggatagccctcaccctgttcaaccttgctgacactctgcttggcggcctgccgacagaattgatttcgtcggctggtggccagctgttctactcccgtcccgttgtctcagccaatggcgagccgactgttaagttgtatacatctgtagagaatgctcagcaggataagggtattgcaatcccgcatgacattgacctcggagaatctcgtgtggttattcaggattatgataaccaacatgaacaagatcggccgacgccttctccagccccatcgcgccctttctctgtccttcgagctaatgatgtgctttggctctctctcaccgctgccgagtatgaccagtccacttatggctcttcgactggcccagtttatgtttctgactctgtgaccttggttaatgttgcgaccggcgcgcaggccgttgcccggtcgctcgattggaccaaggtcacacttgacggtcgccccctctccaccatccagcagtactcgaagaccttctttgtcctgccgctccgcggtaagctctctttctgggaggcaggcacaactaaagccgggtacccttataattataacaccactgctagcgaccaactgcttgtcgagaatgccgccgggcaccgggtcgctatttccacttacaccactagcctgggtgctggtcccgtctccatttctgcggttgccgttttagccccccactctgcgctagcattgcttgaggataccttggactaccctgcccgcgcccatacttttgatgatttctgcccagagtgccgcccccttggccttcagggctgcgctttccagtctactgtcgctgagcttcagcgccttaagatgaaggtgggaaatcgggagttgtag
Hepatitis E virus Pakistan strain (M80581) ORF2:
Atgcgccctcggcctattttgctgttgctcctcatgtttctgcctatgctgcccgcgccaccgcccggtcagccgtctggccgccgtcgtgggcggcgcagcggcggttccggcggtggtttctggggtgaccgggttgattctcagcccttcgcaatcccctatattcatccaaccaaccccttcgcccccgatgtcaccgctgcggccggggctggacctcgtgttcgccaacccgcccgaccactcggctccgcttggcgtgaccaggcccagcgccccgccgctgcctcacgtcgtagacctaccacagctggggccgcgccgctaaccgcggtcgctccggcccatgacaccccgccagtgcctgatgttgactcccgcggcgccatcctgcgccggcagtataacctatcaacatctcccctcacctcttccgtggccaccggcaaatttggttctttacgccgctcctcttagcccgcttctacccctccaggacggcaccaatactcatataatggctacagaagcttctaattatgcccagtaccgggttgctcgtgccacaattcgctaccgcccgctggtccccaacgctgttggtggctacgctatctccatttcgttctggccacagaccaccaccaccccgacgtccgttgacatgaattcaataacctcgacggatgtccgtattttagtccagcccggcatagcctccgagcttgttattccaagtgagcgcctacactatcgcaaccaaggttggcgctctgttgagacctccggggtggcggaggaggaggccacctctggtcttgtcatgctctgcatacatggctcacctgtaaattcttatactaatacaccctataccggtgccctcgggctgttggactttgccctcgaacttgagttccgcaacctcacccccggtaataccaatacgcgggtctcgcgttactccagcactgcccgtcaccgccttcgtcgcggtgcagatgggactgccgagctcaccaccacggctgctactcgcttcatgaaggacctctattttactagtactaatggtgttggtgagatcggccgcgggatagcgcttaccctgtttaaccttgctgacaccctgcttggcggtctaccgacagaattgatttcgtcggctggtggccagctgttctactctcgccccgtcgtctcagccaatggcgagccgactgttaagctgtatacatctgtggagaatgctcagcaggataagggtattgcaatcccgcatgacatcgacctcggggaatcccgtgtagttattcaggattatgacaaccaacatgagcaggaccgaccgacaccttccccagccccatcgcgtcctttttctgtcctccgagctaacgatgtgctttggctttctctcaccgctgccgagtatgaccagtccacttacggctcttcgaccggcccagtctatgtctctgactctgtgaccttggttaatgttgcgaccggcgcgcaggccgttgcccggtcactcgactggaccaaggtcacacttgatggtcgccccctttccaccatccagcagtattcaaagaccttctttgtcctgccgctccgcggtaagctctccttttgggaggcaggaactactaaagccgggtacccttataattataacaccactgctagtgaccaactgctcgttgagaatgccgctgggcatcgggttgctatttccacctacactactagcctgggtgctggccccgtctctatttccgcggttgctgttttagccccccactctgtgctagcattgcttgaggataccatggactaccctgcccgcgcccatactttcgatgacttctgcccggagtgccgcccccttggcctccagggttgtgcttttcagtctactgtcgctgagcttcagcgccttaagatgaaggtgggtaaaactcgggagttatag
Hepatitis E virus Morocco strain (AY230202) ORF2:
Atgcgccctcggcctattttgttgctgttcctcatgtttctgcctatgttgcccgcgccaccgtccggtcagccgtctggccgccgtcgtgggcggcgcagcggcggttccggcggtggtttctggggtgaccgggttgattctcagcccttcgcaatcccctatattcatccaaccaaccccttcgcccccgatgtcaccgctgcggccggggctggacctcgtgttcgccaacccgcccgaccactcggctccgcttggcgtgaccaggcccagcgccccgccgctgcctcacgtcgtagacctaccacagctggggccgcgccgctgactgcggttgctccggcccatgataccccgcctgtgcccgatgttgattctcgcggcgctatcttgcgccggcagtataatctttcaacatcccctcttacctcctccgtggccactggcaccaatttggtcctttatgctgcccctcttagcccgcttttgccccttcaggacggcaccaatactcatataatggccacggaggcttccaattatgcccagtaccgagttgcccgcgccacaatccgctaccgccctctggttcccaatgctgttggtggctatgctatctctatatcgttctggccgcagaccactaccaccccgacgtccgtcgacatgaattctataacctcgacggatgttcgtattctcgtccagcccggtattgcctcagagcttgtgattccaagtgaacgcctgcattatcgcaaccagggttggcgctctgttgaaacctccggggtggcggaggaggaggccacctctggccttgtcatgctttgtatacatggctcacctgtgaactcttatactaacacaccttacactggtgccctcgggttgttggattttgctctcgagcttgagttccgcaacctcacccctggtaacaccaatacacgggtttcccgctactctagcaccgcccgccaccgtcttcgccgtggcgcagacgggactgccgagctcaccaccacggctgcgactcgctttatgaaggatctctatttttctagcactaatggtgttggcgagatcggccgcgggatagcccttaccctgtttaatcttgctgacaccctgcttggcggcctaccgacagaattgatttcgtcggctggtggccaactgttttactctcgcccggtcgtctcagccaatggcgagccgactgtcaagctgtatacatctgtagagaatgctcagcaggataagggtattgcaatccctcacgacatcgaccttggggaatctcgtgtagtcatccaggattatgacaatcagcatgagcaggaccgtccgacaccttccccagccccgtctcgccctttttctgttctccgggccaatgatgtgctttggctctctctcactgctgctgagtatgatcagtccacctatggctcttctactggcccagtctatgtctccgactctgtgaccttggtcaatgttgcgaccggcgcgcaggccgttgctcggtcgcttgactggactaaggtcacacttgatggccgccctctttctactatccagcagtattcgaagactttctttgtcctgccgcttcgtggtaactttccttctgggaggcaggtaccactaaagccgggtacccttacaattataatactactgctagtgaccaactgctcgttgagaatgccgccgggcaccgggtggctatttccacctacaccactagcttgggtgctggccctgtctccatttctgcggtggctgtcctggccccccactctgcgttagcactgcttgaggataccctggattaccctgctcgcgcccatacttttgatgatttctgcccagagtgccgccccctcggcctccagggctgcgcctttcagtccactgtcgctgagctccagcggcttaagatgaaggtgggtaaaactcgggagttatag
Hepatitis E virus Mexico strain (M74506) ORF2:
Atgcgccctaggcctcttttgctgttgttcctcttgtttctgcctatgttgcccgcgccaccgaccggtcagccgtctggccgccgtcgtgggcggcgcagcggcggtaccggcggtggtttctggggtgaccgggttgattctcagcccttcgcaatcccctatattcatccaaccaacccctttgccccagacgttgccgctgcgtccgggtctggacctcgccttcgccaaccagcccggccacttggctccacttggcgagatcaggcccagcgcccctccgctgcctcccgtcgccgacctgccacagccggggctgcggcgctgacggctgtggcgcctgcccatgacacctcacccgtcccggacgttgattctcgcggtgcaattctacgccgccagtataatttgtctacttcacccctgacatcctctgtggcctctggcactaatttagtcctgtatgcagccccccttaatccgcctctgccgctgcaggacggtactaatactcacattatggccacagaggcctccaattatgcacagtaccgggttgcccgcgctactatccgttaccggcccctagtgcctaatgcagttggaggctatgctatatccatttctttctggcctcaaacaaccacaacccctacatctgttgacatgaattccattacttccactgatgtcaggattcttgttcaacctggcatagcatctgaattggtcatcccaagcgagcgccttcactaccgcaatcaaggttggcgctcggttgagacatctggtgttgctgaggaggaagccacctccggtcttgtcatgttatgcatacatggctctccagttaactcctataccaataccccttataccggtgcccttggcttactggactttgccttagagcttgagtttcgcaatctcaccacctgtaacaccaatacacgtgtgtcccgttactccagcactgctcgtcactccgcccgaggggccgacgggactgcggagctgaccacaactgcagccaccaggttcatgaaagatctccactttaccggccttaatggggtaggtgaagtcggccgcgggatagctctaacattacttaaccttgctgacacgctcctcggcgggctcccgacagaattaatttcgtcggctggcgggcaactgttttattcccgcccggttgtctcagccaatggcgagccaaccgtgaagctctatacatcagtggagaatgctcagcaggataagggtgttgctatcccccacgatatcgatcttggtgattcgcgtgtggtcattcaggattatgacaaccagcatgagcaggatcggcccaccccgtcgcctgcgccatctcggcctttttctgttctccgagcaaatgatgtactttggctgtccctcactgcagccgagtatgaccagtccacttacgggtcgtcaactggcccggtttatatctcggacagcgtgactttggtgaatgttgcgactggcgcgcaggccgtagcccgatcgcttgactggtccaaagtcaccctcgacgggcggcccctcccgactgttgagcaatattccaagacattctttgtgctcccccttcgtggcaagctctccttttgggaggccggcacaacaaaagcaggttatccttataattataatactactgctagtgaccagattctgattgaaaatgctgccggccatcgggtcgccatttcaacctataccaccaggcttggggccggtccggtcgccatttctgcggccgcggttttggctccacgctccgccctggctctgctggaggatacttttgattatccggggcgggcgcacacatttgatgacttctgccctgaatgccgcgctttaggcctccagggttgtgctttccagtcaactgtcgctgagctccagcgccttaagttaaggtgggtaaaactcgggagttgtag
Hepatitis E virus U.S. strain (AF060668) ORF2:
Atgcgccctagggctgttctgttgttgttcctcatgtttctgcctatgctgcccgcgccaccggccggtcagccgtctggccgtcgccgtgggcggcgcagcggcggtgccggcggtggtttctggagtgacagggttgattctcagcccttcgccctcccctatattcatccaaccaaccccttcgccgccgatgtcgtttcacaacccggggctggaactcgccctcgacagccgccccgccccctcggttccgcttggcgtgaccagtccaagcgcccctccgttgccccccgtcgtcgatctaccccagctggggctgcgccgctaactgccatatcaccagcccctgatacagctcctgtacctgatgttgactcacgtggtgctattttgcgccggcagtacaatttgtctacgtccccgcttacatcatctgttgcttctggtactaatctggttctctatgctgccccgctgaaccctctcttgcctcttcaggatggcaccaacactcatattatggctactgaggcatctaattacgcccagtatcgggttgttcgggctacgattcgttatcgcccgttggtgccaaatgctgttggtggttatgctatctctatttctttctggcctcaaactacaactacccctacttctgttgacatgaattctatcacttctactgatgtcaggatcttggtccagcccggtatagcctccgagttagtcatccctagtgaacgccttcactaccgcaaccaaggctggcgctctgttgagaccacgggtgtggccgaagaggaggctacctccggtctggtaatgctttgtattcatggctcccctgttaactcctacactaatacaccttacaccggtgcattggggcttcttgattttgcattagaacttgaatttagaaatttgacacccgggaacactaacacccgtgtttcccggtatactagcacagcccgccaccggctgcgccgcggtgctgatgggaccgctgagctcaccaccacagcagccacacgcttcatgaaggatttgcattttactggtacgaacggcgttggtgaggtgggtcgtggtattgccctgactctgtttaatcttgctgatacgcttcttggtggtttaccgacagaattgatttcgtcggctgggggtcaactgttttactcccgccctgttgtctcggccaatggcgagccaacagtaaagttatacacatctgttgagaatgcgcagcaagacaagggcatcaccattccacacgacatagatttaggtgactcccgtgtggttatccaggattatgataaccagcacgaacaagatcgacctaccccgtcacctgccccctcccgccctttctcagttcttcgtgccaatgatgttttgtggctctctctcactgccgctgagtacgrccagaccacgtatgggtcgtccaccaaccctatgtatgtctctgatacagtcacgcttgttaatgtagccactggtgctcaggctgttgcccgctctcttgactggtctaaagttactctggatggtcgccctcttactaccattcagcagtattctaagaaattttatgttctcccgcttcgsgggaagctgtccttttgggaggctggtacgaccaaggccggctacccgtataattataataccactgctagtgaccaaattttgattgagaacgcggccggtcaccgtgtcgccatttctacttataccactagtttgggtgccggccctacctcgatytctgcggtcggtgtactagctccacattcggcccttgctgttctcgaggatactgttgattatcctgctcgtgcccatacttttgatgatttctgcccggagtgtcgcacccttggtctgcagggttgtgcattccaatctactattgctgaacttcagcgtcttaaaatgaaggtaggtaaaacccgggagtcttaa
Hepatitis E virus New Zealand pig strain (AF082843) ORF2:
atgcgccctagggctgttctgttgttgctcttcgtgcttctgcctatgctgcccgcgccaccggccggccagccgtctggccgccgttgtgggcggcgcaacggcggtgccggcggtggtttctggggtgacagggttgattctcagcccttcgccctcccctatattcatccaaccaaccccttcgctgccgatgtcgtttcacaacccggggctggagttcgccctcgacagccgccccgcccccttggctccgcttggcgtgaccagtcccagcgcccctccactgccccccgtcgtcgatctgccccagctggggctgcgccgctgactgctgtatcaccggcccccgacacagctcctgtacctgatgttgactcacgtggtgctatcctgcgccggcagtacaatctgtctacgtccccgctcacgtcatctgtcgctgctggtaccaacctggttctctatgccgccccgctgaatcctctcttgcccctccaggatggcaccaacactcatattatggctactgaggcgtccaattatgctcagtatcgggttgttcgagctacgatccgttatcgcccgctggtgccaaatgctgttggtggctatgctatctctatttctttctggcctcaaactacaaccacccctacttcagttgacatgaactctattacctccactgatgtcaggattttggttcagcccggtattgcctccgagttagtcatccctagtgagcgccttcattaccgcaatcaaggctggcgctctgtagagaccacgggcgtggccgaggaggaagctacctccggtctggtaatgctttgcattcacggttctcctgttaactcctatactaacacaccttacactggtgcattggggctccttgattttgcattagagcttgaattcagaaatttgacacccgggaacactaacacccgtgtttcccggtacaccagcacagcccgccatcggctgcgccgcggtgctgatgggaccgcagagcttaccaccacagcagccacacgtttcatgaaggacttgcatttcaccggcacgaacggcgttggtgaggtgggtcgcggtatagctctaacactgtttaaccttgctgatacgcttcttggtggtttaccgacagaattgatttcgtcggccgggggccaactgttttactcccgccctgtcgtctcggccaatggcgagccgacggttaagttatatacatctgttgagaatgcgcagcaggacaagggcattaccatcccacacgatatagatctgggtgattcccgtgtggttattcaggattatgataaccagcacgagcaagaccgacctactccgtcaccagccccctctcgccctttctcagttcttcgcgccaatgatgttctgtggctctccctcaccgccgctgagtacgatcagactacatatgggtcgtccaccaaccctatgtatgtctccgatacggtcacgctagttaatgtggccactggtgctcaggctgttgcccgctctcttgattggtctaaagtcactctggatggccgccccctcactaccattcagcagtattcaaagacattctatgttctcccgctccgcgggaagctgtccttttgggaggctggtaccactaaggccggctacccgtataattataataccactgctagtgatcaaattttgattgagaacgcggctggccaccgtgttgctatctctacctataccactagcttgggtgccggccctacctcgatttccgccgttggtgtgctagccccacactcggctctcgccgtccttgaggatactgttgattaccctgctcgtgctcatacttttgatgatttctgcccggagtgccgcacccttggtttgcagggttgtgcattccagtctactattgctgagcttcagcgtcttaaaatgaaggtaggtaaaacccgggagtcttaa
Hepatitis E virus Chinese strain (AJ272108) ORF2:
atgaataacatgttcttttgctccgtgcatggagatgccaccatgcgctctcgggctcttctgtttctgctcttcgtgcttctgcctatgttgcccgcgccaccggccggtcagccgtctggccgtcgccgcgggcaagcggggtgcggcggtggtttctggggtgaccgggttgattctcagcccttcgccctcccctatattcatccaaccaaccccttcgcatctgacattccagccgccgccgggactggagctcgccctcggcagccaatccgtccactcggctccgcttggcgtgaccagtcccagcgccccgccgcttccacccgtcgtcgacctgccccagctggggcttcgccgttgactgctgtggctccggctcctgacactgcacctgtccccgatgctgattcccgtggcgctattctacgccgccagtacaatttgtccacatccccgctcacgtccactattgcaactggcaccaattttgtgctatatgctgccccacttagtcccctgctaccgcttcaggatggcaccaatacccacatcatggctactgaagcatctaattatgcccagtatcgtgttgttcgtgctaccatccgatatcgccccttggtgccgaatgccgttggcgggtatgccatatctatctccttttggcctcagacaacgaccaccccaacgtctgttgatatgaattcaattacctccactgatgtccgcattcttgtccagcctggtatagcttctgagttggtaactcccagtgagcgcctgcattatcgaaatcaaggatggcgttcggttgagacctctggtgtcgctgaggaagaagcgacctctggccttgttatgctttgcattcatgggtcacctgtgaattcctatactaatacaccttataccggtgccctcggcttacttgactttgcactcgagctcgagtttcgcaatttgacacctggtaacaccaatacgcgcgtttctcgttattcgagtagcgcgcgtcacaaactgcgccgagggcctgatggtactgctgagttgactactactgccgccacacgttttatgaaagacctccattttacggggactaatggtgttggtgaggtcggtcgtggtatagcgttaactttgtttaatcttgctgatacgcttctcggtgggcttccgacagaattgatttcgtcggcagggggtcagttattctactcccgccccgttgtctcagccaatggcgagctgacagtgaaactttacacttcagtcgagaacgctcagcaggacaagggtgtagctattccacatgatattgaccttggtgagtcccgtgtggttattcaggattatgacaaccaacatgagcaagatcgtcccactccctcccctgctccctctcgtccattttctgttcttcgtgctaatgatgtgctttggctttcacttactgctgctgagtatgatcaaacgacttatggctcctctaccaaccctatgtatgtttctgacactgttacatttgttaacgtagcgactggtgcccagggggtttcgcgctctctggattggtctaaggtcactctcgatggccgtccactcaccaccatccagcagtattctaagactttctatgtcttgccccttcgtggtaagctttccttttgggaggccggcaccactaaagccggctacccttataattataacactactgctagtgaccagatcctgattgagaatgcagcgggccaccgagtttgcatctctacctacactactaacctgggctccgggcctgtgtctgtatctgctgttggtgtcctcgcccctcactctgcgctggctgctttggaggataccgctgactaccctgctcgcgcccatacttttgatgatttctgccctgaatgccgtgcactcggccttcagggttgtgccttccaatctactgttggtgagttacagcgtcttaaaatgaaggtgggtaaaacccgggagtattga
The amino acid sequence of 7 strain hepatitis E virus ORF2 encoding proteins is respectively:
Hepatitis E virus Burma strain (M 73218) pORF2:
MRPRPILLLLLMFLPMLPAPPPGQPSGRRRGRRSGGSGGGFWGDRVDSQPFAIPYIHPTNPFAPDVTAAAGAGPRVRQPARPLGSAWRDQAQRPAVASRRRPTTAGAAPLTAVAPAHDTPPVPDVDSRGAILRRQYNLSTSPLTSSVATGTNLVLYAAPLSPLLPLQDGTNTHIMATEASNYAQYRVARATIRYRPLVPNAVGGYAISISFWPQTTTTPTSVDMNSITSTDVRILVQPGIASELVIPSERLHYRNQGWRSVETSGVAEEEATSGLVMLCIHGSLVNSYTNTPYTGALGLLDFALELEFRNLTPGNTNTRVSRYSSTARHRLRRGADGTAELTTTAATRFMKDLYFTSTNGVGEIGRGIALTLFNLADTLLGGLPTELISSAGGQLFYSRPVVSANGEPTVKLYTSVENAQQDKGIAIPHDIDLGESRVVIQDYDNQHEQDRPTPSPAPSRPFSVLRANDVLWLSLTAAEYDQSTYGSSTGPVYVSDSVTLVNVATGAQAVARSLDWTKVTLDGRPLSTIQQYSKTFFVLPLRGKLSFWEAGTTKAGYPYNYNTTASDQLLVENAAGHRVAISTYTTSLGAGPVSISAVAVLAPHSALALLEDTLDYPARAHTFDDFCPECRPLGLQGCAFQSTVAELQRLKMKVGKTREL.
Hepatitis E virus Pakistan strain (M80581) pORF2:
MRPRPILLLLLMFLPMLPAPPPGQPSGRRRGRRSGGSGGGFWGDRVDSQPFAIPYIHPTNPFAPDVTAAAGAGPRVRQPARPLGSAWRDQAQRPAAASRRRPTTAGAAPLTAVAPAHDTPPVPDVDSRGAILRRQYNLSTSPLTSSVATGTNLVLYAAPLSPLLPLQDGTNTHIMATEASNYAQYRVARATIRYRPLVPNAVGGYAISISFWPQTTTTPTSVDMNSITSTDVRILVQPGIASELVIPSERLHYRNQGWRSVETSGVAEEEATSGLVMLCIHGSPVNSYTNTPYTGALGLLDFALELEFRNLTPGNTNTRVSRYSSTARHRLRRGADGTAELTTTAATRFMKDLYFTSTNGVGEIGRGIALTLFNLADTLLGGLPTELISSAGGQLFYSRPVVSANGEPTVKLYTSVENAQQDKGIAIPHDIDLGESRVVIQDYDNQHEQDRPTPSPAPSRPFSVLRANDVLWLSLTAAEYDQSTYGSSTGPVYVSDSVTLVNVATGAQAVARSLDWTKVTLDGRPLSTIQQYSKTFFVLPLRGKLSFWEAGTTKAGYPYNYNTTASDQLLVENAAGHRVAISTYTTSLGAGPVSISAVAVLAPHSVLALLEDTMDYPARAHTFDDFCPECRPLGLQGCAFQSTVAELQRLKMKVGKTREL.
Hepatitis E virus Morocco strain (AY230202) pORF2:
MRPRPILLLFLMFLPMLPAPPSGQPSGRRRGRRSGGSGGGFWGDRVDSQPFAIPYIHPTNPFAPDVTAAAGAGPRVRQPARPLGSAWRDQAQRPAAASRRRPTTAGAAPLTAVAPAHDTPPVPDVDSRGAILRRQYNLSTSPLTSSVATGTNLVLYAAPLSPLLPLQDGTNTHIMATEASNYAQYRVARATIRYRPLVPNAVGGYAISISFWPQTTTTPTSVDMNSITSTDVRILVQPGIASELVIPSERLHYRNQGWRSVETSGVAEEEATSGLVMLCIHGSPVNSYTNTPYTGALGLLDFALELEFRNLTPGNTNTRVSRYSSTARHRLRRGADGTAELTTTAATRFMKDLYFSSTNGVGEIGRGIALTLFNLADTLLGGLPTELISSAGGQLFYSRPVVSANGEPTVKLYTSVENAQQDKGIAIPHDIDLGESRVVIQDYDNQHEQDRPTPSPAPSRPFSVLRANDVLWLSLTAAEYDQSTYGSSTGPVYVSDSVTLVNVATGAQAVARSLDWTKVTLDGRPLSTIQQYSKTFFVLPLRGKLSFWEAGTTKAGYPYNYNTTASDQLLVENAAGHRVAISTYTTSLGAGPVSISAVAVLAPHSALALLEDTLDYPARAHTFDDFCPECRPLGLQGCAFQSTVAELQRLKMKVGKTREL.
Hepatitis E virus Mexico strain (M74506) pORF2:
MRPRPLLLLFLLFLPMLPAPPTGQPSGRRRGRRSGGTGGGFWGDRVDSQPFAIPYIHPTNPFAPDVAAASGSGPRLRQPARPLGSTWRDQAQRPSAASRRRPATAGAAALTAVAPAHDTSPVPDVDSRGAILRRQYNLSTSPLTSSVASGTNLVLYAAPLNPPLPLQDGTNTHIMATEASNYAQYRVARATIRYRPLVPNAVGGYAISISFWPQTTTTPTSVDMNSITSTDVRILVQPGIASELVIPSERLHYRNQGWRSVETSGVAEEEATSGLVMLCIHGSPVNSYTNTPYTGALGLLDFALELEFRNLTTCNTNTRVSRYSSTARHSARGADGTAELTTTAATRFMKDLHFTGLNGVGEVGRGIALTLLNLADTLLGGLPTELISSAGGQLFYSRPVVSANGEPTVKLYTSVENAQQDKGVAIPHDIDLGDSRVVIQDYDNQHEQDRPTPSPAPSRPFSVLRANDVLWLSLTAAEYDQSTYGSSTGPVYISDSVTLVNVATGAQAVARSLDWSKVTLDGRPLPTVEQYSKTFFVLPLRGKLSFWEAGTTKAGYPYNYNTTASDQILIENAAGHRVAISTYTTRLGAGPVAISAAAVLAPRSALALLEDTFDYPGRAHTFDDFCPECRALGLQGCAFQSTVAELQRLKVKVGKTREL.
Hepatitis E virus U.S. strain (AF060668) pORF2:
MRPRAVLLLFLMFLPMLPAPPAGQPSGRRRGRRSGGAGGGFWSDRVDSQPFALPYIHPTNPFAADVVSQPGAGTRPRQPPRPLGSAWRDQSKRPSVAPRRRSTPAGAAPLTAISPAPDTAPVPDVDSRGAILRRQYNLSTSPLTSSVASGTNLVLYAAPLNPLLPLQDGTNTHIMATEASNYAQYRVVRATIRYRPLVPNAVGGYAISISFWPQTTTTPTSVDMNSITSTDVRILVQPGIASELVIPSERLHYRNQGWRSVETTGVAEEEATSGLVMLCIHGSPVNSYTNTPYTGALGLLDFALELEFRNLTPGNTNTRVSRYTSTARHRLRRGADGTAELTTTAATRFMKDLHFTGTNGVGEVGRGIALTLFNLADTLLGGLPTELISSAGGQLFYSRPVVSANGEPTVKLYTSVENAQQDKGITIPHDIDLGDSRVVIQDYDNQHEQDRPTPSPAPSRPFSVLRANDVLWLSLTAAEYXQTTYGSSTNPMYVSDTVTLVNVATGAQAVARSLDWSKVTLDGRPLTTIQQYSKKFYVLPLRGKLSFWEAGTTKAGYPYNYNTTASDQILIENAAGHRVAISTYTTSLGAGPTSISAVGVLAPHSALAVLEDTVDYPARAHTFDDFCPECRTLGLQGCAFQSTIAELQRLKMKVGKTRES.
Hepatitis E virus New Zealand pig strain (AF082843) pORF2:
MRPRAVLLLLFVLLPMLPAPPAGQPSGRRCGRRNGGAGGGFWGDRVDSQPFALPYIHPTNPFAADVVSQPGAGVRPRQPPRPLGSAWRDQSQRPSTAPRRRSAPAGAAPLTAVSPAPDTAPVPDVDSRGAILRRQYNLSTSPLTSSVAAGTNLVLYAAPLNPLLPLQDGTNTHIMATEASNYAQYRWRATIRYRPLVPNAVGGYAISISFWPQTTTTPTSVDMNSITSTDVRILVQPGIASELVIPSERLHYRNQGWRSVETTGVAEEEATSGLVMLCIHGSPVNSYTNTPYTGALGLLDFALELEFRNLTPGNTNTRVSRYTSTARHRLRRGADGTAELTTTAATRFMKDLHFTGTNGVGEVGRGIALTLFNLADTLLGGLPTELISSAGGQLFYSRPVVSANGEPTVKLYTSVENAQQDKGITIPHDIDLGDSRVVIQDYDNQHEQDRPTPSPAPSRPFSVLRANDVLWLSLTAAEYDQTTYGSSTNPMYVSDTVTLVNVATGAQAVARSLDWSKVTLDGRPLTTIQQYSKTFYVLPLRGKLSFWEAGTTKAGYPYNYNTTASDQILIENAAGHRVAISTYTTSLGAGPTSISAVGVLAPHSALAVLEDTVDYPARAHTFDDFCPECRTLGLQGCAFQSTIAELQRLKMKVGKTRES.
Hepatitis E virus Chinese strain (AJ272108) pORF2:
MNNMFFCSVHGDATMRSRALLFLLFVLLPMLPAPPAGQPSGRRRGQAGCGGGFWGDRVDSQPFALPYIHPTNPFASDIPAAAGTGARPRQPIRPLGSAWRDQSQRPAASTRRRPAPAGASPLTAVAPAPDTAPVPDADSRGAILRRQYNLSTSPLTSTIATGTNFVLYAAPLSPLLPLQDGTNTHIMATEASNYAQYRVVRATIRYRPLVPNAVGGYAISISFWPQTTTTPTSVDMNSITSTDVRILVQPGIASELVTPSERLHYRNQGWRSVETSGVAEEEATSGLVMLCIHGSPVNSYTNTPYTGALGLLDFALELEFRNLTPGNTNTRVSRYSSSARHKLRRGPDGTAELTTTAATRFMKDLHFTGTNGVGEVGRGIALTLFNLADTLLGGLPTELISSAGGQLFYSRPVVSANGELTVKLYTSVENAQQDKGVAIPHDIDLGESRVVIQDYDNQHEQDRPTPSPAPSRPFSVLRANDVLWLSLTAAEYDQTTYGSSTNPMYVSDTVTFVNVATGAQGVSRSLDWSKVTLDGRPLTTIQQYSKTFYVLPLRGKLSFWEAGTTKAGYPYNYNTTASDQILIENAAGHRVCISTYTTNLGSGPVSVSAVGVLAPHSALAALEDTADYPARAHTFDDFCPECRALGLQGCAFQSTVGELQRLKMKVGKTREY.
Antigen protein is selected the recombinant protein or the polypeptide of hepatitis E virus ORF2 or its fragment coding, preferred hepatitis E virus ORF2 166 the amino acid whose recombinant proteins between the 452nd～617 amino acids of encoding, and called after p166, amino acid sequence is as follows:
Hepatitis E virus Burma strain (M 73218) p166Bur:
TPSPAPSRPFSVLRANDVLWLSLTAAEYDQSTYGSSTGPVYVSDSVTLVNVATGAQAVARSLDWTKVTLDGRPLSTIQQYSKTFFVLPLRGKLSFWEAGTTKAGYPYNYNTTASDQLLVENAAGHRVAISTYTTSLGAGPVSISAVAVLAPHSALALLEDTLDYP
Hepatitis E virus Pakistan strain (M80581) p166Sar:
PTPSPAPSRPFSVLRANDVLWLSLTAAEYDQSTYGSSTGPVYVSDSVTLVNVATGAQAVARSLDWTKVTLDGRPLSTIQQYSKTFFVLPLRGKLSFWEAGTTKAGYPYNYNTTASDQLLVENAAGHRVAISTYTTSLGAGPVSISAVAVLAPHSVLALLEDTMDYP
Hepatitis E virus Morocco strain (AY230202) p166Mor:
PTPSPAPSRPFSVLRANDVLWLSLTAAEYDQSTYGSSTGPVYVSDSVTLVNVATGAQAVARSLDWTKVTLDGRPLSTIQQYSKTFFVLPLRGKLSFWEAGTTKAGYPYNYNTTASDQLLVENAAGHRVAISTYTTSLGAGPVSISAVAVLAPHSALALLEDTLDYP
Hepatitis E virus Mexico strain (M74506) p166Mex:
PTPSPAPSRPFSVLRANDVLWLSLTAAEYDQSTYGSSTGPVYISDSVTLVNVATGAQAVARSLDWSKVTLDGRPLPTVEQYSKTFFVLPLRGKLSFWEAGTTKAGYPYNYNTTASDQILIENAAGHRVAISTYTTRLGAGPVAISAAAVLAPRSALALLEDTFDYP
Hepatitis E virus U.S. strain (AF060668) p166Us:
PTPSPAPSRPFSVLRANDVLWLSLTAAEYXQTTYGSSTNPMYVSDTVTLVNVATGAQAVARSLDWSKVTLDGRPLTTIQQYSKKFYVLPLRGKLSFWEAGTTKAGYPYNYNTTASDQILIENAAGHRVAISTYTTSLGAGPTSISAVGVLAPHSALAVLEDTVDYP
Hepatitis E virus New Zealand pig strain (AF082843) p166Nz:
PSPSPAPSRPFSVLRANDVLWLSLTAAEYDQTTYGSSTNPMYVSDTVTFVNVATGAQAVARSLDWSKVTLDGRPLTTIQQYSKTFYVLPLRGKLSFWEAGTTKAGYPYNYNTTASDQILIENAAGHRVAISTYTTSLGAGPVSVSAVGVLAPHSALAILEDTVDYS
Hepatitis E virus Chinese strain (AJ272108) p166Chn:
PTPSPAPSRPFSVLRANDVLWLSLTAAEYDQTTYGSSTNPMYVSDTVTFVNVATGAQGVSRSLDWSKVTLDGRPLTTIQQYSKTFYVLPLRGKLSFWEAGTTKAGYPYNYNTTASDQILIENAAGHRVCISTYTTNLGSGPVSISAVGVLAPHSALAALEDTVDYP
(1) according to the synthetic Auele Specific Primer of sequence, classifies template as, the genetic fragment of above-mentioned 7 strain hepatitis E virus coding ORF2C end, 452～617 amino acids that increase respectively with above-mentioned 7 strain hepatitis E virus genome sequences.The PCR condition is: 94 ℃-45 seconds, 52 ℃-45 seconds, 72 ℃-50 seconds, 35 circulations.The PCR product reclaims purifying through Ago-Gel, is cloned into plasmid vector pGEX-4T-2 carrier (Pharmacia product).
Primer sequence is:
Hepatitis E virus Burma strain (M 73218) p166Bur: primer B1:CCC GGA TTC CCG ACGCCT TCT CCA GCC CCA TC, primer B2:CCC CTC GAG TCA AGG GTA GTCCAA GGT ATC CTC AA;
Hepatitis E virus Pakistan strain (M80581) p166Sar: primer S1:CCC GGA TTC CCG ACCCCT TCT CCT GCC CCA TC, primer S2:CCC CTC GAG TCA AGG GTA GTC CATGGT ATC CTC AA;
Hepatitis E virus Morocco strain (AY230202) p166Mor: primer Mo1:CCC GGA TTC CCGACC CCT TCT CCT GCC CCA TC, primer Mo2:CCC CTC GAG TCA AGG GTAATC CAG GGT ATC CTC AA
Hepatitis E virus Mexico strain (M74506) p166Mex: primer Me1:CCC GGA TTC CCG ACCCCT TCT CCT GCC CCA TC, primer Me2:CCC CTC GAG TCA CGG ATA ATCAAA AGT ATC CTC CA;
Hepatitis E virus U.S. strain (AF060668) p166Us: primer U1:CCC GGA TTC CCG ACCCCT TCT CCT GCC CCATC, primer U2:CCC CTC GAG TCA TCC TAT TAG TTGTCA TAG GAG C:
Hepatitis E virus New Zealand pig strain (AF082843) p166Nz: primer N1:CCC GGA TTC CCGACC CCT TCT CCT GCC CCA TC, primer N2:CCC CTC GAG TCA CGA ATA ATCAAC AGT ATC TTC AA;
Hepatitis E virus Chinese strain (AJ272108) p166Chn: primer C1:CCC GGA TTC CCG ACCCCT TCT CCT GCC CCA TC, primer C2:CCC CTC GAG TCA AGG GTA ATC AACAGT GTC CTC CA.
(2) expression plasmid is transformed in the expression strain E.coli JM109 bacterial strain (Promega product), picking list bacterium colony is with containing the LB medium culture of 50ug/ml ampicillin to OD
550=0.6～0.8, with final concentration is that 0.2～1.0mM IPTG induces, inducing temperature is 37 ℃, shaking table shakes speed and is 200rpm, centrifugal collection thalline after 3-4 hour, with cell pyrolysis liquid (50mM Tris-HCl, pH 7.2,300mM NaCl) suspension bacterial sediment, ultrasonication thalline after the freeze thawing 6 times, centrifugal collection supernatant is through GST-Sepharose4B (Pharmacia product) affinitive layer purification, obtaining 7 kinds, to represent the recombinant protein of hepatitis E virus different genotype and hypotype be antigen, respectively called after p166Bur, p166Pak, p166Mor, p166Mex, p166Us, p166Nz and p166Chn.Use UV at last
280, method such as SDS-PAGE detects, quantitatively.
2, the preparation of neutralizing monoclonal antibody and evaluation
(1) animal immune: adopt conventional method each 50 μ g of recombinant protein p166Bur, p166Mex, p166US and p166Chn and freund adjuvant mixing lumbar injection immunity female Balb/c mouse in 6-8 age in week to represent 4 kinds of oligogene type hepatitis E virus respectively, totally 3 times, 2 thoughtful January at interval.
(2) foundation of hybridoma cell strain: use immune p166Bur, p166Mex, p166US and p166Chn recombinant protein bag by enzyme reaction plate, the GST bag is by enzyme reaction plate hole in contrast.The screening of employing indirect elisa method, all p166Bur, p166Mex, p166US and the p166Chn positive, the GST negative patient is the positive hole of monoclonal antibody simultaneously.Through 3 subclones, the hybridoma cell strain that obtains the stably excreting monoclonal antibody corresponds to 4G5,8G10,3D4 and 5G5 respectively.
(3) a large amount of preparations and the evaluation of monoclonal antibody: adopt preparation ascites and the upright cultivation of culture flask to prepare monoclonal antibody in a large number.With the rabbit anti-mouse igg subclass antibody of HRP mark and the anti-mouse IgM of rabbit antibody is two anti-, adopts indirect elisa method to detect and identifies monoclonal antibody immunity globulin type and subclass.Collected culture supernatant or ascites detect antibody titer with the ELISA method behind doubling dilution.
The immunoglobulin class of table 1. hepatitis E virus monoclonal antibody and subclass and antibody titer thereof
| 4G5 | 8G10 | 3D4 | 5G5 | |
| Ig class and subclass antibody titer | IgG1 1∶10 -5 | IgG2b 1∶10 -5 | IgG1 1∶10 -5 | Ig2b 1∶10 -5 |
(4) the monoclonal antibody specificity is identified:
The reactivity of the different hepatitis E virus genotype of monoclonal antibody s that employing indirect elisa method and the evaluation of Western-blot method are obtained and the p166 recombinant protein (p166Bur, p166Pak, p166Mor, p166Mex, p166Us, p166Nz and p166Chn) of hypotype with 7 kinds.
The reactivity of table 2. hepatitis E virus monoclonal antibody and different genotype and hypotype p166 recombinant protein
| Monoclonal antibody | Different genotype and hypotype p166 antigen ELISA detect the P/N value of different monoclonal antibodies | |||||||
| Bur (Ia) | Pak (Ib) | Mor (Ic) | Mex (II) | US (III) | Nz (III) | Chn (IV) | GST | |
| 4G5 8G10 3D4 5G5 | 25.9 22.0 11.2 14.2 | 22.1 20.0 12.5 15.5 | 21.9 18.2 10.02 13.8 | 19.7 22.4 10.8 14.9 | 18.3 24.4 13.1 19.6 | 14.4 24.2 12.7 18.5 | 12.8 23.0 11.4 22.3 | 0.13 0.61 0.65 0.48 |
(5) the external neutralization test of monoclonal antibody is identified:
1) cultivates the PLC/PRF/5 cell and grow to individual layer;
2) with the monoclonal antibody ascites 100ul of doubling dilution with contain 100TCID
50/ 100ul viral hepatitis type E viral suspension mixes, and hatches inoculation PLC/PRF/5 cell after 2 hours jointly for 37 ℃;
3) 37 ℃ hatch 2 hours jointly after, the virus that flush away does not adsorb, use the Trizol method to extract nucleic acid, with primer 3 (5 '-CCG ACA GAA TTG ATT TCG TCG GC-3 '), primer 4 (5 '-GTT GTC TCGGCC AAT GGC GAG CC-3 '), primer 5 (5 '-TCG GCG GCG GTG AGA GAG AGCCA-3 '), primer 6 (5 '-CCG TAA GTG GAC TGG TCG TAC TC-3 ') carries out reverse transcription-nest-type PRC and detects hepatitis E virus nucleic acid, the nest-type PRC condition is: the first round: 94 ℃-45 seconds, 50 ℃-45 seconds, 72 ℃-50 seconds, 35 circulations, primer are primer 3 and primer 6; Second takes turns: 94 ℃-45 seconds, 52 ℃-45 seconds, 72 ℃-50 seconds, 30 circulations, primer is primer 4 and primer 5;
4) the RT-PCR feminine gender is judged to the neutralization test positive, and monoclonal antibody 4G5,8G10,3D4 and the 5G5 of the virus that will neutralize at last are judged to the hepatitis E virus neutralizing monoclonal antibody.
(6) Purification of Monoclonal Antibodies:
1) the centrifugal removal precipitation of monoclonal antibody, filter paper filtering is removed lipid, adds the 1M Tris.HCl of 1/10 volume, and PH 8.0, regulate PH to neutral;
2) with the 1MTris.HCl of 10 times of volumes, the Protein G Ago-Gel prepacked column that PH 8.0 balance Pierce companies produce;
3) go up sample: get the pretreated monoclonal antibody ascites of 1ml upper prop, incubated at room 10min;
4) wash post: (the 0.1M Tris.HCl, PH 8.0) that add 10 times of volumes washes post, and flush away is the albumen of combination not, and detection A280 reduces to baseline to the A value;
5) wash-out: add the elution buffer 0.05M Gly.HCl of 1 times of column volume, the monoclonal antibody of PH 2.5-3.0 elution of bound on purification column collected, mixed at every turn, and it is quantitative to measure the A280 value ,-20 ℃ of preservations after the packing.
3, enzyme labeling MONOCLONAL ANTIBODIES SPECIFIC FOR
Present embodiment is that example describes the enzyme labeling MONOCLONAL ANTIBODIES SPECIFIC FOR in detail with the horseradish peroxidase.
1) takes by weighing horseradish peroxidase 5mg, be dissolved in the 0.2ml deionized water, drip freshly prepared 0.1mol/L sodium metaperiodate (NaIO
4) solution 0.04ml, stirring at room 20 minutes;
2) horseradish peroxidase of hydroformylation is packed into bag filter is to 1mmol/L acetate buffer solution (PH4.4) dialysed overnight;
3) adding 0.2mol/L carbonate buffer solution (PH9.5) adjustment horseradish peroxidase solution pH value is 9.0～9.5, adds hepatitis E virus neutralizing monoclonal antibody (PH9.0～9.5) immediately;
4) add freshly prepared sodium borohydride solution (4mg/ml) 0.02ml, 4 ℃ static 2 hours;
5) add the saturated metabisulfite solution of equivalent, 4 ℃ static 30 minutes, centrifugal 30 minutes of 5000rpm removes supernatant;
6) precipitation is with an amount of PBS dissolving, and the bag filter of packing into to PBS dialysis 48 hours, is the hepatitis E virus neutralizing monoclonal antibody of horseradish co-oxidation thing enzyme labeling, adds 1% bovine serum albumin(BSA), adds equal-volume glycerine again ,-20 ℃ of preservations.
4, the foundation of hepatitis E virus neutralizing antibody competitive ELISA detection method
As envelope antigen, can form envelope antigen with the recombinant protein of hepatitis E virus ORF2 or its fragment coding or polypeptide alone or in combination, be the foundation that example describes hepatitis E virus neutralizing antibody competitive ELISA detection method in detail with the p166 recombinant protein below.
(1) envelope antigen:
1) envelope antigen can be selected like this: recombinant protein p166Bur, p166Pak, p166Mor, p166Mex, p166Us, p166Nz and the p166Chn of above-mentioned 7 strain hepatitis E virus ORF2C end coding, 452～617 amino acids can form envelope antigen respectively, be diluted to 10～2000ng/ml with the PH9.50.05mol/L carbonate buffer solution, 100 μ l/ holes add enzyme reaction plate, and 4 ℃ of bags are spent the night.
2) envelope antigen also can be selected like this: recombinant protein p166Bur, p166Pak, p166Mor, p166Mex, p166Us, p166Nz and the p166Chn of above-mentioned 7 strain hepatitis E virus ORF2C end coding, 452～617 amino acids can mix by 1: 1 ratio isoconcentration, be diluted to 10～2000ng/ml with the PH9.50.05mol/L carbonate buffer solution again, 100 μ l/ holes add enzyme reaction plate, and 4 ℃ of bags are spent the night.
(2) washing: bag was by second day, and (PH7.00.01mol/L PBS 0.05%tween-20) washes plate 3 times with cleansing solution;
(3) detect: get bag by reaction plate, adding sample to be tested 50 μ l, positive control hole add and add known negative serum 50 μ l in known positive serum 50 μ l, the negative control hole in detecting the hole, the reference control wells adds PBS (0.05mol/L, PH7.0) 50 μ l, add enzyme mark hepatitis E virus neutralizing monoclonal antibody 50 μ l again, hatched jointly 40 minutes for 37 ℃, cleansing solution is washed plate 5 times, the unconjugated antibody of flush away;
(4) colour developing: add the substrate of enzyme, this substrate can be the substrate of horseradish peroxidase, and 10～15min develops the color under the room temperature;
(5) stop: add the reaction of stop buffer color development stopping, read the OD value;
(6) result judges: according to formula inhibiting rate=(1-testing sample hole OD value/reference control wells OD value) * 100%, be judged as the positive with inhibiting rate greater than 50%.
5, the application of hepatitis E virus neutralizing antibody competitive ELISA detection method in the hepatitis E virus vaccine is estimated
Gather the rhesus macaque serum of the hepatitis E virus ORF2 recombinant protein vaccine immunity of this laboratory preparation, wherein 10 μ g group, 20 μ g group are each 3, per 4 all immunity once, immunity is 3 times altogether, 3 of control group rhesus macaquies adopt same approach with PBS (.0.05mol/3, PH7.0) 1ml immunity, per 4 all immunity once are total to immunity 3 times.Experimental group and 9 rhesus macaquies of control group are with Chinese strain hepatitis E virus viral suspension 1ml (PCR titre 10 during 8 weeks
7) attack.Since each organize rhesus macaque took a blood sample once weekly in 0 week, uses hepatitis E virus neutralizing antibody competitive ELISA detection method of the present invention and detect the neutrality antibody production, sees Fig. 1.From figure data as seen, immune group is from immunity 4 week beginnings, serum is anti--the hepatitis E virus neutralizing antibody has obvious rising, and peak beginning neutralizing antibody competition inhibiting rate 〉=50% in about 6 weeks in 8-9 after week.Control group from virus attack 4 week the back serum anti--the hepatitis E virus neutralizing antibody begins to raise, and peaks after attacking malicious 9 weeks.To prove that hepatitis E virus neutralizing antibody competitive ELISA that we set up detects anti--and the hepatitis E virus neutralizing antibody is effective, can be used for the screening and the evaluation of vaccine.
Claims (2)
1, a kind of competitive enzyme-linked immune detection method of hepatitis E virus neutralizing antibody, it is characterized in that serum (slurry) or other biological imitate anti-hepatitis E virus neutralizing antibody and enzyme labeling hepatitis E virus neutralizing monoclonal antibody in this compete combine on the envelope antigen in and epitope, according to the degree that the zymolyte chromogenic reaction weakens, realize the competitive enzyme-linked immune of the hepatitis E virus neutralizing antibody in the biological sample is detected.
2, the competitive enzyme-linked immune detection method of hepatitis E virus neutralizing antibody as claimed in claim 1, envelope antigen is the hepatitis E virus open reading frame 2 of different genotype and hypotype or the recombinant protein or the polypeptide of its fragment coding, can form envelope antigen alone or in combination.
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