CN101699291A - Method for determining cross antigen region initiating human antigens in human enterovirus 71-type total protein - Google Patents
Method for determining cross antigen region initiating human antigens in human enterovirus 71-type total protein Download PDFInfo
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Abstract
The invention belongs to the field of biomedicine, and provides a method for determining a cross antigen region initiating human antigens in human enterovirus 71-type (EV71) total protein. In the method, the human enterovirus 71-type total protein is identified through segmental expression; and cross reaction between immunogenic and inductive antibodies and the human antigens in each segment is performed, and different segments of the human enterovirus 71-type total protein are divided into three classes: (1) strongly-crossed immunogens initiating the human antigens; (2) weakly-crossed immunogens initiating the human antigens; and (3) no crossed immunogens existing between the antibodies and the human antigens. Thus, the method plays a guidance role in developing and preparing human enterovirus 71-type vaccines, and hand-foot-and-mouth disease vaccines having no cross reaction or weak cross reaction with human bodies can be designed according to the method.
Description
Technical field
The invention belongs to biomedical sector, particularly relate to the method for research human enterovirus 71.
Background technology
Human enterovirus 71 (EV71) is separated first evaluation in 1969, is one of pathogen of hand-foot-and-mouth disease.This virus mainly infects children below 5 years old, mainly causes hand-foot-and-mouth disease and herpangina, and the complication under a few cases comprises meningitis, and pulmonary edema is hemorrhage etc., and serious case can cause death.Human enterovirus 71 caused once that worldwide repeatedly outbreak of disease was popular, and since the eighties in last century, the hand-foot-and-mouth disease that human enterovirus 71 causes is wreaked havoc in the Asian-Pacific area, has caused serious infant's death.
China found this disease from 1981 in Shanghai, and all there is report in each provinces and cities later on.In March, 2009, the whole nation was 54713 examples by the hand-foot-and-mouth disease case of the straight reporting system report of infectious disease network, dead 31 examples.The beginning of the year, network was directly reported demonstration to April 7 from this year, and whole nation accumulative total reports the hand-foot-and-mouth disease example 115618 examples, severe 773 examples wherein, dead 50 examples.
2,193 amino acid of human enterovirus 71 polypeptied chain total length cut into 11 albumen after synthesizing in vivo, comprising: VP1, VP2, VP3, VP4,2A, 2B, 2C, 3A, 3B, 3C, 3D.
Comparatively popular human enterovirus 71 mechanism of causing a disease is at present, behind the virus infections human body, cause that the blood-brain barrier perviousness increases, the virus massive duplication of neuronal cell and cellular damage of initiation in brain subsequently, caused neuronal pulmonary edema and pulmonary edema, this is serious case of infant and main causes of death.
But, comparative medicine in mouse body test simultaneously shows, the morbidity of newborn rat does not have the massive duplication of associated virus at brain tissue, traditional viral pathogenesis cognition has been proposed new challenge, determining the pathogenesis that human enterovirus 71 infects, is hand-foot-and-mouth disease treatment and the important prerequisite of preventing.
Summary of the invention
Need in view of above, fundamental purpose of the present invention is to provide the method for determining to cause in the human enterovirus 71 total protein human antigen's the former zone of cross immunity.
In order to reach above purpose, cause the method in human antigen's the former zone of cross immunity in definite human enterovirus 71 total protein provided by the invention, it mainly may further comprise the steps:
A. express cutting apart of human enterovirus 71 total protein with engineering
The human enterovirus 71 polypeptied chain is divided into the polypeptide of being made up of 22～156 amino acid, totally 22,, uses 6 * His tag label to carry out purifying respectively at expression in escherichia coli;
B. the human enterovirus 71 total protein cuts apart determining of polypeptide antigen
The albumen of purifying is carried out immunity to animal used as test, the antiserum that obtains carries out the SABC test with the brain tissue of normal adults and fetus respectively, cross reaction can take place with normal cerebral tissue and get antiserum in evaluation, has determined to exist with human body the human enterovirus 71 polypeptide in the former zone of strong cross immunity; Determined to exist the human enterovirus 71 polypeptide in the weak former zone of cross immunity with human body; Determined not exist the human enterovirus 71 polypeptide in the former zone of cross immunity with human body.
Among the above-mentioned steps a, the polypeptied chain of EV71 virus is carried out order cut apart, 2193 amino acid are divided into the polypeptide that 22～156 amino acid constitute successively from the N end, totally 22, the molecular weight of most polypeptide is about 10kDa;
Among the above-mentioned steps a, adjacent and belong to exist between two polypeptide of an albumen 4～19 amino acid whose overlapping, avoided the screening of antigenic determinant to omit;
Among the above-mentioned steps a, the N of polypeptide end carries out amalgamation and expression with 6 * His tag label, because 6 * His tag albumen label can not produce obviously influence to the antigenicity of polypeptide, helps the purifying of polypeptide simultaneously;
Among the above-mentioned steps b, polypeptide is to the animal used as test immunity, and described animal used as test comprises rat, mouse, rabbit, monkey etc.;
Among the above-mentioned steps b, P
230-323, P
646-755, P
857-1012, P
1329-14404 peptide species and human antigen's cross reaction result is a strong positive, and index number is illustrated in polypeptide position on the polypeptied chain in total virus, and initial from the N end, they belong to the VP2 in the human enterovirus 71 respectively, VP1,2A, 2C albumen; P
1-69, P
324-443, P
444-565, P
566-665, P
746-876, P
1441-1526, P
1549-1668, P
1732-1851, P
1952-2071, P
2072-219310 peptide species and human antigen's cross reaction result is the weak positive, and they belong to the VP4 in the human enterovirus 71 respectively, VP3, VP1,3A, 3C and 3D albumen; P
70-159, P
140-249, P
1197-1338, P
1649-1731, P
1843-1951There are not cross reaction in 5 peptide species and human antigen, and they belong to the VP2 in the human enterovirus 71 respectively, 2C, 3C and 3D albumen.
Can learn in sum, there are cross-immune reaction in EV71 antiserum and human antigen, and determined to cause in the EV71 total protein human antigen's the former zone of cross immunity, the different segments with the human enterovirus 71 total protein are divided into 3 classes in view of the above: (1) initiation human antigen's strong cross immunity is former; (2) initiation human antigen's weak cross immunity is former; (3) do not exist cross immunity former with the human antigen.Therefore, the present invention will have directive function to the development of human enterovirus 71 vaccine, can be according to the present invention the vaccine for hand-foot-mouth disease of design and human body no cross reaction or weak cross reaction, wherein, do not exist the former zone of cross immunity can be used for designing vaccine with the human antigen in the human enterovirus 71 total protein, the vaccine for hand-foot-mouth disease of preparation and human body no cross reaction.Exist the former zone of weak cross immunity can be used for designing vaccine with the human antigen in the human enterovirus 71 total protein, the vaccine for hand-foot-mouth disease of preparation and the weak cross reaction of human body.
Embodiment
Below in conjunction with specific embodiments the present invention is described in further detail, but its qualification of not opposing:
Implementation step one
Cut apart and the engineering of human enterovirus 71 total protein are expressed
Cut apart the human enterovirus 71 separated strain that the template that is relied on is Fuyang, Chinese Anhui Province, Genbank storage number is EU703812.The genome C DS district of this bacterial strain is 6582bp altogether, 2193 amino acid of encoding, and the size of cutting apart polypeptide is controlled at about 100 amino acid (aa), under be limited to 22aa, on be limited to 156aa, be positioned between adjacent two polypeptide of same albumen and have the overlapping of 4～15aa, as table 1.
The partitioning scheme of table 1 human enterovirus 71 total protein
NdeI and SacI site are added in the primer two ends of amplification polypeptid coding area, expression vector uses transformed pET28a (+) expression vector, this carrier is through after revising, and the N end of expressed destination protein does not contain unnecessary amino acid sequence except that 6 * His tag albumen label.
The expression vector that successfully constructs changes among the Escherichia coli BL21 (DE3), induces expression of polypeptides with 0.5mM IPTG.Expression of polypeptides detects with SDS-PAGE, and the result shows except that P
1013-1111, P
1112-1201, P
1527-1548Outside three expression of polypeptides failures, all the other polypeptide are successful expression all.
Implementation step two
The purifying of cutting apart polypeptide
Polypeptide is with insoluble inclusion body formal representation, and the inclusion body of purifying uses the dissolving of 8M urea, uses the Ni-HTA of Novagen company to carry out affinitive layer purification subsequently, and polypeptide purity reaches more than 95% behind the purifying.
Implementation step three
Determine to have the former polypeptide of cross immunity with the human antigen
Polypeptide behind the purifying is to the ICR mouse immune, and the antiserum of acquisition carries out the SABC test with the brain tissue of normal adults and fetus respectively, determines that human antigen's cross immunity is former.
The antiserum extension rate is 1: 200, and two anti-sides are two anti-available from the anti-mouse of HRP labelled goat of middle mountain company, and chromogenic substrate is DAB, uses the observation by light microscope test findings subsequently.The result shows, with human antigen's cross reaction be strong positive comprise EV71 total virus and P
230-323, P
646-755, P
857-1012, P
1329-14404 peptide species.With human antigen's cross reaction be the p+ P that comprises
1-69, P
324-443, P
444-565, P
566-665, P
746-876, P
1441-1526, P
1549-1668, P
1732-1851, P
1952-2071, P
2072-219310 peptide species.The P that is that does not have cross reaction with the human antigen
70-159, P
140-249, P
1197-1338, P
1649-1731, P
1843-19515 peptide species.
The above results shows, by expressing cutting apart of human enterovirus 71 total protein with engineering, can determine to have the former zone of immunological cross-reaction with the human antigen in the virus, can be according to the present invention the vaccine for hand-foot-mouth disease of design and human body no cross reaction or weak cross reaction.
Below its content of the present invention has been done to elaborate.For persons skilled in the art, any conspicuous change of under the prerequisite that does not deviate from the principle of the invention it being done can not exceed the protection domain of the application's claims.
Claims (7)
1. determine to cause in the human enterovirus 71 total protein method in human antigen's the former zone of cross immunity, it is characterized in that, mainly may further comprise the steps:
A. express cutting apart of human enterovirus 71 total protein with engineering
The human enterovirus 71 polypeptied chain is divided into the polypeptide of being made up of 22～156 amino acid from N end order successively, totally 22,, uses 6 * His tag label to carry out purifying respectively at expression in escherichia coli;
B. the human enterovirus 71 total protein cuts apart determining of polypeptide antigen
The polypeptide of purifying is carried out immunity to animal used as test, the antiserum that obtains carries out the SABC test with the brain tissue of normal adults and fetus respectively, the antiserum of cross reaction can take place with normal cerebral tissue in evaluation, has determined to exist with human body the human enterovirus 71 polypeptide in the former zone of strong cross immunity; Determined to exist the human enterovirus 71 polypeptide in the weak former zone of cross immunity with human body; Determined not exist the human enterovirus 71 polypeptide in the former zone of cross immunity with human body.
2. cause the method in human antigen's the former zone of cross immunity in definite human enterovirus 71 total protein according to claim 1, it is characterized in that, among the described step a, cut apart in 22 polypeptide of generation, be positioned at exist between the adjacent polypeptide of same albumen 4～19 amino acid whose overlapping.
3. cause the method in human antigen's the former zone of cross immunity in definite human enterovirus 71 total protein according to claim 1, it is characterized in that among the described step b, polypeptide is to the animal used as test immunity, described animal used as test comprises rat, mouse, rabbit, monkey.
4. cause the method in human antigen's the former zone of cross immunity in definite human enterovirus 71 total protein according to claim 1, it is characterized in that, among the described step b, cause human antigen's former comprising of strong cross immunity: P
230-323, P
646-755, P
857-1012And P
1329-1440, wherein, described index number is illustrated in polypeptide position on the polypeptied chain in total virus, and initial from the N end, they belong to the VP2 in the human enterovirus 71 respectively, VP1,2A, 2C albumen.
5. cause the method in human antigen's the former zone of cross immunity in definite human enterovirus 71 total protein according to claim 1, it is characterized in that, among the described step b, cause human antigen's former comprising of weak cross immunity: P
1-69, P
324-443, P
444-565, P
566-665, P
746-876, P
1441-1526, P
1549-1668, P
1732-1851, P
1952-2071And P
2072-2193, wherein, described index number is illustrated in polypeptide position on the polypeptied chain in total virus, and initial from the N end, they belong to the VP4 in the human enterovirus 71 respectively, VP3, VP1,3A, 3C and 3D albumen.
6. cause the method in human antigen's the former zone of cross immunity in definite human enterovirus 71 total protein according to claim 1, it is characterized in that, among the described step b, do not have with the human antigen that cross immunity is former to be comprised: P
70-159, P
140-249, P
1197-1338, P
1649-1731, P
1843-1951, wherein, described index number is illustrated in polypeptide position on the polypeptied chain in total virus, and initial from the N end, they belong to the VP2 in the human enterovirus 71 respectively, 2C, 3C and 3D albumen.
7. cause the method in human antigen's the former zone of cross immunity in definite human enterovirus 71 total protein as claimed in claim 1, be used to design or the vaccine for hand-foot-mouth disease of preparation and human body no cross reaction or weak cross reaction.
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Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103387615A (en) * | 2012-05-09 | 2013-11-13 | 北京工业大学 | Hand-foot-and-mouth disease vaccine based on HBc and EV71 VP4, and preparation method and application thereof |
| CN104945481A (en) * | 2014-12-31 | 2015-09-30 | 苏州偲聚生物材料有限公司 | Polypeptide, detection device comprising polypeptide, and detection kit comprising device |
| CN104945478A (en) * | 2014-12-31 | 2015-09-30 | 苏州偲聚生物材料有限公司 | Polypeptide, detection device comprising polypeptide, and detection kit comprising device |
| CN104945477A (en) * | 2014-12-31 | 2015-09-30 | 苏州九龙医院有限公司 | Polypeptide, detection device comprising polypeptide, and detection kit comprising device |
| CN104945479A (en) * | 2014-12-31 | 2015-09-30 | 苏州偲聚生物材料有限公司 | Polypeptide, detection device comprising polypeptide, and detection kit comprising device |
| CN105085672A (en) * | 2015-01-19 | 2015-11-25 | 鄢慧民 | 3D protein specific monoclonal immunoglobulin A antibodies and composition thereof |
| CN105732769A (en) * | 2014-12-10 | 2016-07-06 | 苏州偲聚生物材料有限公司 | Polypeptides, detection device comprising the polypeptides, and detection kit comprising the detection device |
| CN105732770A (en) * | 2014-12-10 | 2016-07-06 | 苏州偲聚生物材料有限公司 | Polypeptides, a detection device comprising the polypeptides, and a detection kit comprising the detection device |
| WO2016115665A1 (en) * | 2015-01-19 | 2016-07-28 | Huimin Yan | Immunoglobulin g monoclonal antibodies against 3d proteins of enteroviruses |
| CN108267577A (en) * | 2018-01-15 | 2018-07-10 | 广州市妇女儿童医疗中心 | A kind of EV71 viruses IgA antibody test strip |
| CN108912214A (en) * | 2015-11-27 | 2018-11-30 | 山东大学 | The immunogenic polypeptide and the preparation method and application thereof of enterovirns type 71 VP1 antigen |
| CN112194709A (en) * | 2020-12-07 | 2021-01-08 | 北京纳百生物科技有限公司 | Immunochromatographic test strip for detecting feline panleukopenia syndrome virus antibody, and preparation method and application thereof |
| CN113150131A (en) * | 2021-02-26 | 2021-07-23 | 上海市公共卫生临床中心 | Monoclonal antibody for broad-spectrum recognition of group A enterovirus 2C protein and application thereof |
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Cited By (15)
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| CN103387615B (en) * | 2012-05-09 | 2015-07-22 | 北京工业大学 | Hand-foot-and-mouth disease vaccine based on HBc and EV71 VP4, and preparation method and application thereof |
| CN103387615A (en) * | 2012-05-09 | 2013-11-13 | 北京工业大学 | Hand-foot-and-mouth disease vaccine based on HBc and EV71 VP4, and preparation method and application thereof |
| CN105732770A (en) * | 2014-12-10 | 2016-07-06 | 苏州偲聚生物材料有限公司 | Polypeptides, a detection device comprising the polypeptides, and a detection kit comprising the detection device |
| CN105732769A (en) * | 2014-12-10 | 2016-07-06 | 苏州偲聚生物材料有限公司 | Polypeptides, detection device comprising the polypeptides, and detection kit comprising the detection device |
| CN104945481A (en) * | 2014-12-31 | 2015-09-30 | 苏州偲聚生物材料有限公司 | Polypeptide, detection device comprising polypeptide, and detection kit comprising device |
| CN104945478A (en) * | 2014-12-31 | 2015-09-30 | 苏州偲聚生物材料有限公司 | Polypeptide, detection device comprising polypeptide, and detection kit comprising device |
| CN104945477A (en) * | 2014-12-31 | 2015-09-30 | 苏州九龙医院有限公司 | Polypeptide, detection device comprising polypeptide, and detection kit comprising device |
| CN104945479A (en) * | 2014-12-31 | 2015-09-30 | 苏州偲聚生物材料有限公司 | Polypeptide, detection device comprising polypeptide, and detection kit comprising device |
| CN105085672A (en) * | 2015-01-19 | 2015-11-25 | 鄢慧民 | 3D protein specific monoclonal immunoglobulin A antibodies and composition thereof |
| WO2016115665A1 (en) * | 2015-01-19 | 2016-07-28 | Huimin Yan | Immunoglobulin g monoclonal antibodies against 3d proteins of enteroviruses |
| CN108912214A (en) * | 2015-11-27 | 2018-11-30 | 山东大学 | The immunogenic polypeptide and the preparation method and application thereof of enterovirns type 71 VP1 antigen |
| CN108912214B (en) * | 2015-11-27 | 2021-07-16 | 山东大学 | Immunogenic polypeptide of enterovirus 71 type VP1 antigen and preparation method and application thereof |
| CN108267577A (en) * | 2018-01-15 | 2018-07-10 | 广州市妇女儿童医疗中心 | A kind of EV71 viruses IgA antibody test strip |
| CN112194709A (en) * | 2020-12-07 | 2021-01-08 | 北京纳百生物科技有限公司 | Immunochromatographic test strip for detecting feline panleukopenia syndrome virus antibody, and preparation method and application thereof |
| CN113150131A (en) * | 2021-02-26 | 2021-07-23 | 上海市公共卫生临床中心 | Monoclonal antibody for broad-spectrum recognition of group A enterovirus 2C protein and application thereof |
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Application publication date: 20100428 |