CN101710119A - Establishment and application of detection method for in vitro evaluation of influenza A virus subtype H1N1 antibody titer - Google Patents
Establishment and application of detection method for in vitro evaluation of influenza A virus subtype H1N1 antibody titer Download PDFInfo
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- CN101710119A CN101710119A CN200910272207A CN200910272207A CN101710119A CN 101710119 A CN101710119 A CN 101710119A CN 200910272207 A CN200910272207 A CN 200910272207A CN 200910272207 A CN200910272207 A CN 200910272207A CN 101710119 A CN101710119 A CN 101710119A
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Abstract
The invention belongs to the field of research on zoonosis, and relates to a differential diagnosis method for zoonosis. The method comprises: selecting a HA gene in a cDNA sequence of disclosed influenza H1N1 California virus strain (A/California/08/2009 (H1N1)) as research content; analyzing the sequence of the part of HA protein leaking out of peplos according to structural biology software; selecting the part with higher immunogenicity according to antigenicity analysis; obtaining a gene fragment by a gene synthesis method to build a prokaryotic expression vector pGEX-6P-1-HA; transforming positive recombinant into escherichia coli to obtain recombinant strain (Escherichia Coli BL21 Rosseta/pGEX-6P-1-HA truncated protein); obtaining HA truncated protein of purified H1N1 by a plurality of chromatography methods; and utilizing indirect-method experimental principles of expression protein establishment enzyme-linked immunosorbent assay to evaluate the antibody titer produced inside patients because of influenza virus infection or vaccine immunization.
Description
Technical field
The invention belongs to the zoonosis research field, relate to a kind of antidiastole method of zoonosis.Selected that the HA gene is a research contents in disclosed H1N1 californian virus strain (A/California/08/2009 (H1N1)) the cDNA sequence, at first go out the sequence that HA albumen leaks part outside coating according to the structure biology software analysis, again according to antigenicity analysis, select immunogenicity than higher part, obtain genetic fragment by method for synthesizing gene and make up prokaryotic expression carrier pGEX-6P-1-HA; Positive recombinant plasmid transformed is obtained recombinant bacterial strain (Escherichia coli BL21rosseta/pGEX-6P-1-HA truncated protein) in Escherichia coli; By the HA truncated protein of the H1N1 behind the multiple chromatography method acquisition purifying, and utilize described expressing protein to set up in the enzyme linked immunosorbent assay indirect method experimental principle evaluation patient body because the antibody titer that influenza infection or vaccine immunity produce.
The invention provides the expression and purification method of the strong pathogenic important epitope albumen of H1N1virus HA of reorganization and with the diagnostic method of the H1N1 antibody of this albumen foundation.
Background technology
Influenza A H1N1 is a kind of respiratory diseases in pigs that is caused by A type swine influenza virus, and this virus can cause influenza to break out in swinery.In by the end of March, 2009 to mid-April, the multinational Type A Influenza H1N1 epidemic situations of breaking out in succession such as Mexico, the U.S., then in a few short months the time at whole world rapid spread.This virus that causes epidemic situation is swine influenza virus A (H1N1) hypotype, is a kind of novel swine influenza virus that never occurred on one's body people and pig.By on September 3rd, 2009, epidemic situation took place in 188 countries and regions, the whole world, reports 250,000 cases altogether, accumulative total death 2800 many cases, and in fact number of the infected will be considerably beyond the cases reported number.At present, the Northern Hemisphere enters autumn, and epidemic situation is fast rise trend.In China, some new variations have also taken place in the epidemic situation situation in the recent period, and epidemic situation to the whole nation, spread to the rural area from the city, is that native country be main by being input as main transformer from coastal, by Sporadic cases to assembling the epidemic situation development.At present, epidemic situation is all found in 31 provinces, interior ground, and reported cases every day number is in rising trend, has also occurred severe cases in the recent period successively.The epidemic situation prevention and control situation that we face allows of no optimist.By September 10,31 the province accumulative totals in ground reported the Influenza A H1N1 confirmed cases 6968 examples in China.
In the latent period of Influenza A H1N1, more common influenza, bird flu are long latent period, and latent period, duration was seven days.Early symptom is similar to ordinary people's influenza, comprises heating, cough, laryngalgia, physical distress, headache etc., and some diarrhoea or vomiting, courbature or tired, symptom such as eyes are rubescent also can occur.The part conditions of patients can make progress rapidly, break with tremendous force, suddenly high heat, body temperature is above 39 ℃, even the serious pneumonia of secondary, acute respiratory distress syndrome, empsyxis, pleural effusion, the minimizing of whole body haemocyte, renal failure, septicemia, shock and Reye syndrome, respiratory failure and multiple organ injury, cause death.Spreading to population health and socio-economic development of Influenza A H1N1 brings significant damage.After epidemic situation was broken out, China paid much attention to the control prevention and control, crisp decision making, and a series of in order effective prevention and control measures have been taked in unified plan.Autumn and winter season, Influenza A H1N1 can enter a peak period.School starts to school becomes the important risk factor of aggregation morbidity.So the situation is tense for China prevention and control first type H1NI influenza.A few days ago, State Council to autumn and winter season China's first type H1NI influenza prevention and control made a series of important deployment.The prevention and control policy of next stage is: strengthen preventive measure, the community that keeps under strict control propagates, and strengthens the severe treatment, reduces epidemic situation harm with all strength.So-called " community ", emphasis comprises school, hospital etc.China formulates the perfect information issue mechanism at the railway under the intensive mobility status of personnel, civil aviaton's prevention and control prediction scheme.Simultaneously, give full play to the vaccine means, particularly in the personnel that participate in the parade on National Day, at first start vaccine inoculation.The first batch of Influenza A H1N1 vaccine of China formally rolled off the production line June 22, and after process a series of biologies, biochemical test and clinical trial, this batch vaccine is expected to listing in September.The consumption of 5,000,000 person-portions was satisfied output in expectation before National Day, and will all be used for official reserves.At present, China has become that first can use the country of Influenza A H1N1 vaccine in the world.
The vaccine inoculation work expansion just in full preparation of nationwide, but some queries are also following.These queries are primarily aimed at the effect whether first stream vaccine " a pin immunity " can reach actual immunity, the aspects such as persistence of immunity.In the news briefing of holding on September 8th, 2009, the director Zhang Wei of medicine registration department of State Food and Drug Administration represents just tentative at present inoculation one pin when responding the query of relevant first stream vaccine " a pin immunity ".First stream vaccine inoculation one pin is enough on earth, produce effective antibody? how is the persistence of immunity? the numerous common people appoint and so have such worry.Have do not have a kind of fast, easy method is made assessment accurately to immune effect of vaccine?
The kit that our company developed can be quick, accurate, cheap the detection antibody titer, effectively solved this difficult problem.
Summary of the invention
The preparation method of wherein said recombinant protein, it comprises the following steps:
1) the HA gene is a genes of interest in disclosed H1N1 californian virus strain (A/California/08/2009 (H1N1)) the cDNA sequence, and the method synthetic by gene obtains genetic fragment;
2) the directed dna sequence dna that inserts H1N1virus nonstructural protein gene HA truncated protein in prokaryotic expression carrier pGEX-6p-1 multiple clone site makes up and obtains prokaryotic expression carrier plasmid pGEX-6p-1-HA truncated protein gene;
3) with step 2) said prokaryotic expression carrier plasmid pGEX-6p-1-HA truncated protein is converted into e. coli bl21 Rosseta competent cell, with the single reorganization of ammonia benzyl mycin resistance screening bacterium.
4) described purifying specifically: the pGEX-6p-1-HA truncated protein is expressed in Escherichia coli Rosetta, and the clone is seeded to the LB damping fluid that 3ml contains the 100ug/ml ampicillin, 37 ℃, spends the night 220rpm; Overnight culture is diluted to the LB damping fluid that 1L contains the 100ug/ml ampicillin at 1: 100, and 37 ℃, 220rpm cultivates 5h; Adding final concentration is the IPTG of 0.1mM, and 22 ℃, 180rpm continues to cultivate 22h; With 8000g, 15min, 4 ℃ of centrifugal receipts bacterium; Thalline 50mM Tris-cl, 200mM NaCl pH8.0 has hanged to 25ml, adds the 5mg lysozyme, RT, 0.5h; Ultrasonic with Ultrasonic Cell Disruptor to solution clarification, 12000rpm, centrifugal 3 times of 20min; Supernatant is used the Glutathione-Sepharose4B post material purifying of GE healthcare, obtain purified fusion protein.The PSP enzyme is cut the GST label, post material purifying, the HA truncated protein of the label that is removed.
HA truncated protein solubility is high after experiment showed, purifying, does not have polymkeric substance to occur in 1 year.
The Pgex-6p-1 of utilization provided by the invention system amalgamation and expression is cut to handle through enzyme again and is removed the HA truncated protein that label makes, can be in Escherichia coli successful expression, expression and forefathers' result is similar, with respect to eukaryotic expression, prokaryotic expression preparation engineering antibody technique is simple relatively, and cost is lower;
Two of purpose of the present invention provides a kind of ELISA diagnostic method and diagnostic kit of quick, accurate, safe, cheap detection H1N1virus antibody horizontal.
The concrete grammar of described diagnostic kit preparation:, extract the also important epitope of HA of purifying expression through centrifugal broken BL21rosseta cell.Prepared ELISA antigen ELISA Plate with this albumen bag by the ELISA ELISA Plate.Prepare serum dilution with blank bacterium (Escherichia coli BL21Rosseta/HA) induced product, detect serum to be checked according to common ELISA method.Determine negative, suspicious, positive judgement critical value according to reacted light absorption value.
According to following formulation coating buffer, cleansing solution, confining liquid, dilution, substrate solution, stop buffer:
Coating buffer (25mmol/L carbonate buffer solution): Na
2CO
32.322g, NaHCO
32.352g, ddH
2O adds to 1000mL (pH9.6).
1 times of cleansing solution: NaCl 8g, KCl 0.2g, Na
2HPO
412H
2O 3.63g, KH
2PO
40.44g distilled water adds to 1000mL (pH7.4).
Confining liquid: 2g bovine serum albumin(BSA) (BSA) is dissolved in the 100mL cleansing solution.
Dilution: 1g bovine serum albumin(BSA) (BSA) is dissolved in the 100mL cleansing solution
Substrate solution: buy from day strong biotinylated biomolecule pharmacy (Tianjin) company limited
Stop buffer: 2N H
2SO
4
HA antigen ELISA Plate, cleansing solution (25 times), dilution, the anti-human IgG antibody of HRP mark, substrate buffer solution, stop buffer are assembled into the HA-ELISA differential diagnosis kit.
The invention has the beneficial effects as follows:
1. one of beneficial effect of the present invention is the biological safety height.The used detection antigen of the present invention is recombinant protein, does not relate to live virus in the preparation process, therefore the potential danger that does not have live virus to escape, spread.
2. two of beneficial effect of the present invention is that production cost is low.Influenza A H1N1 antibody detection method provided by the present invention and the required antigen of detection kit are the important epitopes of the HA of H1N1virus.This albumen can obtain a large amount of expression external by recombination bacillus coli BL21Rosseta/HA truncated protein, is fit to large-scale production.
3. the detection kit that three of beneficial effect of the present invention provides is easy to use.The present invention is on the basis that detection method is provided, and also that this method is required all ingredients is assembled into kit, and operation is simple, is fit to very much clinical extensive detection.
Description of drawings
Fig. 1, demonstration Pgex-6P-1 carrier collection of illustrative plates.
Fig. 2, the warm albumen of HA truncated protein are expressed in a small amount and are identified the SDS-PAGE electrophoretogram.Wherein 1 is inducible strain not; M is the molecular weight of albumen standard, is followed successively by 115KD, 66KD, 45KD, 35KD, 25KD, 18.4KD, 14.4KD from top to bottom; 2 is to induce the back bacterial strain for No. 1; 3 is to induce the back bacterial strain for No. 2; 4 is to induce the back bacterial strain for No. 3;
Fig. 3, demonstration be the warm protein SDS-PAGE electrophoretogram of HA truncated protein behind the purifying.Wherein 1 is warm albumen behind the purifying; M is the molecular weight of albumen standard, is followed successively by 115KD, 66KD, 45KD, 35KD, 25KD, 18.4KD, 14.4KD from top to bottom;
After cutting, Fig. 4, enzyme remove the HA truncated protein SDS-PAGE electrophoretogram of label.Wherein 1 for enzyme cut after the HA albumen of tape label not; M is the molecular weight of albumen standard, is followed successively by 115KD, 66KD, 45KD, 35KD, 25KD, 18.4KD, 14.4KD from top to bottom.
The patent embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
The structure of embodiment 1 H1N1virus HA truncated protein gene prokaryotic carrier
BamHI and SalI enzyme are cut PUC57-HA truncated protein and carrier pGEX-6P-1, reclaim HA truncated protein gene and carrier pGEX-6p-1; Connect with T4DNA ligase then, 22 degree water-baths 1 hour, transformed into escherichia coli Rossetta competent cell, 37`C cultivates, therefrom a plurality of single bacterium colonies of random choose are put into the LB fluid nutrient medium respectively and are cultivated after 5 hours for 37 ℃, and thalline OD value added IPTG to final concentration 1mM at about 0.6 o'clock, identify the picking positive colony through expressing in a small amount.
A large amount of abduction deliverings and the protein purification of embodiment 2 H1N1virus HA
The pGEX-6p-1-HA truncated protein is expressed in Escherichia coli Rosetta, and the clone is seeded to the LB damping fluid that 3ml contains the 100ug/ml ampicillin, 37 ℃, spends the night 220rpm; Overnight culture is diluted to the LB damping fluid that 1L contains the 100ug/ml ampicillin at 1: 100, and 37 ℃, 220rpm cultivates 5h; Adding final concentration is the IPTG of 0.1mM, and 22 ℃, 180rpm continues to cultivate 22h; With 8000g, 15min, 4 ℃ of centrifugal receipts bacterium; Thalline 50mM Tris-Cl, 200mM NaCl pH8.0 has hanged to 25ml, adds the 5mg lysozyme, RT, 0.5h; Ultrasonic with Ultrasonic Cell Disruptor to solution clarification, 12000rpm, centrifugal 3 times of 20min; Supernatant is used the Glutathione-Sepharose4B post material purifying of GE healthcare, obtain purified fusion protein.The PSP enzyme is cut the GST label, post material purifying, the HA truncated protein of the label that is removed.
1.1 the microwell plate of envelope antigen (8 holes * 12 * 1)
1.2HRP the anti-human IgG1's bottle of mark (120ul/ bottle)
1.325 1 bottle of cleansing solution concentrate (20ml/ bottle) doubly
1.4 2 bottles of dilutions (20ml/ bottle)
1.5 1 bottle of substrate solution (10ml/ bottle)
1.6 1 bottle of stop buffer (10ml/ bottle)
1) take out kit from 4 ℃ of refrigerators, each component of balance is to room temperature.
2) get pre-bag quilt the micropore batten (according to sample what, removable gradation is used), except that the blank hole, every hole adds the sample to be checked with sample diluting liquid dilution in 1: 40, and every hole adds 100ul, same 1: 40 dilution control serum, if positive control 2 holes, negative control 2 holes, blank well does not add, shake gently and spare sample (not overflowing) in the hole, put 37 ℃ of incubations 1 hour.
3) get rid of solution in the plate hole, wash plate 3 times with cleansing solution, 200ul/ time, leave standstill at every turn and outwelled in 3 minutes, pat dry for the last time.
4) every hole adds ELIAS secondary antibody 100ul, puts 37 ℃ of incubations 30 minutes.
5) washing is 4 times, and 200ul/ time, leave standstill at every turn and outwelled in 3 minutes, pat dry for the last time.
6) every hole adds substrate 90ul, room temperature lucifuge colour developing (in 30 minutes).
7) every hole adds stop buffer 50ul, measurement result in 15 minutes.
Claims (4)
1. an in-vitro evaluation is at the foundation of the detection method of influenza A virus subtype H 1 N 1 antibody titer.
2. H1N1virus according to claim 1, it is characterized in that, include but not limited to part-structure, reorganization H1N1 envelope protein and the reorganization H1N1 non-structural protein of whole virus particles virion of H1N1virus of whole virus particles, the deactivation of live body H1N1virus.
3. antibody according to claim 1, it is characterized in that, include but not limited to that part-structure, reorganization H1N1 envelope protein and the reorganization H1N1 non-structural protein of whole virus particles virion of H1N1virus of whole virus particles, the deactivation of live body H1N1virus enters the immunoglobulin (Ig) of the antigen that produces behind the host by some approach as antigen.
4. detection method according to claim 1 is characterized in that, sets up the method for the antibody titer of anti-H1N1 in the immunological method test sample that includes but not limited to immunoturbidimetry and enzyme linked immunological (ELISA) method based on immunological method.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103116029A (en) * | 2013-01-29 | 2013-05-22 | 福州迈新生物技术开发有限公司 | Determining method for sensitivity and affinity of second antibody color appearance system for immunohistochemistry |
| CN103439515A (en) * | 2013-08-14 | 2013-12-11 | 南方医科大学 | Method for detecting valence of antibody |
-
2009
- 2009-09-25 CN CN200910272207A patent/CN101710119A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103116029A (en) * | 2013-01-29 | 2013-05-22 | 福州迈新生物技术开发有限公司 | Determining method for sensitivity and affinity of second antibody color appearance system for immunohistochemistry |
| CN103116029B (en) * | 2013-01-29 | 2015-01-21 | 福州迈新生物技术开发有限公司 | Determining method for sensitivity and affinity of second antibody color appearance system for immunohistochemistry |
| CN103439515A (en) * | 2013-08-14 | 2013-12-11 | 南方医科大学 | Method for detecting valence of antibody |
| CN103439515B (en) * | 2013-08-14 | 2015-07-15 | 南方医科大学 | Method for detecting valence of antibody |
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Open date: 20100519 |