CN110470829A - A kind of 6 gene of the rotavirus vp that amino acid sequence morphs and immuno-chromatographic test paper strip - Google Patents

A kind of 6 gene of the rotavirus vp that amino acid sequence morphs and immuno-chromatographic test paper strip Download PDF

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CN110470829A
CN110470829A CN201910844679.5A CN201910844679A CN110470829A CN 110470829 A CN110470829 A CN 110470829A CN 201910844679 A CN201910844679 A CN 201910844679A CN 110470829 A CN110470829 A CN 110470829A
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rotavirus
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immuno
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朱传刚
周志平
冒丽
刘冀
江嘉欣
吴思敏
李嘉静
纪荣毅
沈元曦
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Nevonos Suzhou Bioengineering Co ltd
Shanghai Veteromaru Research Institute Caas China Animal Health And Epidemiology Center Shanghan Branch Center
Shanghai Veterinary Research Institute CAAS
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Shanghai Veteromaru Research Institute Caas China Animal Health And Epidemiology Center Shanghan Branch Center
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    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
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    • C12N2720/00011—Details
    • C12N2720/12011—Reoviridae
    • C12N2720/12311—Rotavirus, e.g. rotavirus A
    • C12N2720/12322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

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Abstract

The present invention provides a kind of 6 gene of the rotavirus vp that amino acid sequence morphs and immuno-chromatographic test paper strip, and the rotavirus colloidal gold immuno-chromatography test paper strip is equipped with carrier board, sample pad, gold conjugation pad, laminate film, detection line, nature controlling line and absorption pad.Using recombination VP6 albumen as detection antigen, colloid gold particle is prepared for using most classic trisodium citrate reduction method, the development and pig porcine rotavirus positive serum for completing rotavirus immuno-chromatographic test paper strip can be specifically bound, and tentatively establish the fast diagnosis method of diagnosis rotavirus.

Description

A kind of 6 gene of the rotavirus vp that amino acid sequence morphs and immunity-chromatography test Paper slip
Technical field
The invention belongs to antigen preparation technical fields, and in particular to a kind of antigen of novel Rotavirus Vaccine, i.e., one 6 gene of rotavirus vp that kind amino acid sequence morphs, and the rotavirus colloidal gold containing the antigen quickly detect examination Paper slip and its application.
Background technique
Rotavirus (Rotavirus, RV) belongs to Reoviridae, rotavirus, in 1973 by head such as Bishop Secondary discovery is gained the name rotavirus because it is similar to vehicle wheel spoke under Electronic Speculum.The virus is to cause the mankind, mammal and birds The main pathogens of virus diarrhea, the infant that the people at highest risk of virus infection is -3 years old 6 months, clinical symptoms show as holding The diarrhea and vomiting for continuing more days, along with fever, hematochezia, serious person can be because of complication such as dehydration, toxic myocarditis and pneumonia And it is dead, the grice diarrhoea caused by China is because of porcine rotavirus (Porcine rotavirus) causes seriously to pass through to pig breeding industry Ji loss.
Complete rotaviral particles have three layers of capsid, and genome is made of 11 double-stranded RNA segments, Mei Geji Because segment encodes an albumen, genetic fragment 6 is responsible for coding inner casing configuration albumen VP6.VP6 albumen is that content is most in rotavirus High albumen is group (subgroup) antigen protein of virus, important function is played in the assembling process of virion, in inner casing The assembling of proteinic genome RNA and coat protein, which enter in cell, plays the role of a physics receptor, while VP6 albumen It is essential in the presence of the transcription for virus.After human or animal infects rotavirus, it is (sub- that anti-rotavirus group can be generated in vivo Group) antibody, detect that this antibody can be detected the infection of rotavirus, in view of have strongly immunogenic of VP6 albumen and Well-conserved, VP6 is widely used in the detection of rotavirus.
VP6 albumen accounts for the 51% of rotavirus total protein content, has important biological function, is maintaining viral shape State, cell entry cell, genome duplication and assembling process play an important role.VP6 albumen cannot stimulate in body generation And antibody, but generated IgA has protective effect, and which also has the cytotoxic T of a cross reaction thin (CTL) epitope.So far there are no the specific medicament for the treatment of rotavirus infection, virus can only all be reduced for the vaccine of the virus The death rate and severe diarrhea caused by infection do not have and protect the effect of body is from rotavirus infection.The disease with it is other Bacterium and virus caused by diarrhoeal diseases be clinically difficult to identify, can Accurate Diagnosis to treat the virus infection have Very important meaning.The kit of detection rotavirus is used mainly for clinical detection at present, and is primarily used to detection colyliform Viral antigen, it there is no the kit of detection RV antibody.Therefore, it is necessary to develop a kind of quick and convenient, specific detection wheel The colloidal gold strip of shape antiviral antibody.
But it is more due to rotavirus gene group in rotavirus recombinant antigen subunit vaccine used at present research Sample causes the vaccine of preparation that cannot effectively prevent the rotavirus of different genes background.And rotavirus sero-group and blood Clear type is numerous, and the biological characteristics of different strains are different, therefore separation identification and the research of correlation properties to rotavirus, nothing By being basic research to the virus also or all have highly important meaning to the action oriented research of the disease.
Summary of the invention
It is real the invention mainly solves the technical problem of providing a kind of quick, highly sensitive immune detection product and method Now efficient, highdensity immune detection.
It is an object of the present invention to provide one kind to have antigenic rotavirus structural proteins VP6, recombinant protein The gene order of VP6 is as shown in SEQ ID NO.1.
Further, a kind of rotavirus colloidal gold immune chromatography test prepared using above-mentioned VP6 recombinant protein is provided Item.
The rotavirus colloidal gold immuno-chromatography test paper strip is equipped with carrier board, sample pad, gold conjugation pad, laminate Film, detection line, nature controlling line and absorption pad；The sample pad, gold conjugation pad, laminate film and absorption pad are successively pasted onto carrier One end of plate upper surface, sample pad is located on one end of gold conjugation pad, and the other end of gold conjugation pad is located at laminate film One end on, one end of absorption pad is located on the other end of laminate film, and detection line, nature controlling line are sequentially arranged on laminate film；It is examining VP6 recombinant protein is coated at survey line, coating selects streptococcus recombinant protein (rSPG) at nature controlling line.
PVC board can be used in the carrier board.
Using recombination VP6 albumen as detection antigen, colloidal gold is prepared for using most classic trisodium citrate reduction method Specific knot can occur for particle, the development and pig porcine rotavirus positive serum for completing rotavirus immuno-chromatographic test paper strip It closes, tentatively establishes the fast diagnosis method of diagnosis rotavirus.
Detailed description of the invention
The PCR of Fig. 1 ET-32a (+)-VP6 recombinant plasmid identifies electrophoretogram, 1:DNA Market(DM2000)；2:PCR is produced Object；
The double digestion of Fig. 2 pET-32a (+)-VP6 recombinant plasmid identifies electrophoretogram, 1:DNA Market(DM5000)；2:pET- 32a (+)-VP6 recombinant plasmid；
The inducing expression phase of Fig. 3 Vp6 albumen, 1:Protein Market(12-120kDa)；2: no IPTG induction；3-6: add Enter after IPTG 1h, 2h, 3h, 4h, 6h, 8h after inducing expression；
Fig. 4 VP6 albumen solubility qualification result, 1:Protein Market(12-120kDa)；2: the supernatant after ultrasound；3: super Precipitating after sound；
Fig. 5 VP6 protein purification is as a result, 1:Protein Market(12-120kDa)；2: the destination protein 3 before loading: loading Destination protein 4-8: destination protein after purification afterwards；
Fig. 6 colloidal gold immuno-chromatography test paper strip schematic diagram, wherein 1, sample pad；2, PVC bottom plate；3, colloidal gold pad；4, it tests Line；5, control line；6, nitrocellulose filter (NC film)；7, water absorption pad.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified It is commercially available from routine biochemistry reagent shop.
The building expression and activity identification of 1 rotavirus vp of embodiment, 6 recombination
With reference to the gene order of the GenBank VP6 albumen delivered, building has antigenic gene order, application DNAstar software analyzes amino acid sequence, theoretical molecular weight and isoelectric point expressed by recombination sequence, 5, end addition BamH I (GGATCC) restriction enzyme site, 3, TAA termination sequence and Hind is added in end(GAATTC) restriction enzyme site, recombination sequence by Suzhou Jin Weizhi Biotechnology Co., Ltd is synthesized, and the gene order of VP6 is as shown in SEQ ID NO.1.
VP6 gene order segment and pET-32a (+) empty carrier are attached with T4 ligase, constructed pET-28a (+)- R VP6 recombinant plasmid, the LB in recombinant plasmid transformed to e. coli bl21 (DE3), being coated in that penicillin antibody containing card is solid 37 DEG C of overnight incubations on body culture medium, several single bacterium colonies of picking expand culture later.Carry out bacterium solution PCR(Fig. 1) and matter Grain double digestion (Fig. 2) and sequencing identification, are consistent with expection.PCR the primer is T7 universal primer.
By 5mL sequencing, correctly the bacterial cultures containing pET-32a (+)-VP6 recombinant plasmid is inoculated in 250 mL containing ammonia It in the LB liquid medium of parasiticin, is placed in 37 DEG C of concussion and cultivate cases, 250 rpm shaken cultivations.When Bacteria Culture to OD When being 0.6, the IPTG that concentration is 1mmol/L is added and carries out inducing expression, before taking induction respectively, 1h, 2h after induction, 3h, 4h, The bacterium solution of 6h, 8h each 1mL, 12000rpm are centrifuged 10min, abandon supernatant, and precipitating is resuspended with 1 × PBS of 40 μ L, carry out SDS-PAGE Electrophoresis analyzes best induction time.SDS-PAGE electrophoresis result shows rotavirus Vp6 albumen success table in Escherichia coli It reaches, molecular weight is 34.2kDa(Fig. 3).
Bacterium solution 12000rmp after induction is centrifuged 15min, abandons supernatant, precipitating is resuspended with 1 × PBS of 15mL, is frozen repeatedly Melt 3 times, ultrasonication 20min(ultrasonic time 2s, dead time 9.9s), 12000rmp is centrifuged 15min, collects supernatant respectively Precipitating.Precipitating is resuspended with the 8moL urea of 10mL, and 12000rmp is centrifuged 15min, collects supernatant.SDS-PAGE electrophoretic analysis expression The solubility (Fig. 4) of product.It is purified using recombinant protein of the His affinity column to expression, collects each section of eluent sample Product (Fig. 5).
The preparation of 2 colloidal gold immuno-chromatography test paper strip of embodiment
2.1, protein expression: preparation expression, purification of recombinant proteins VP6 as described in Example 1.
2.2, the point sample of nitrocellulose filter: being diluted to 0.5mg/ml for VP6, is used for the scribing line of detection line (T line), will be small The anti-His tag monoclonal antibody of mouse is diluted to scribing line of the 0.5mg/ml for nature controlling line (C line).NC film is affixed on bottom plate, it It is crossed afterwards with colloidal gold spotting system, scribing line amount is 1 μ l/cm, and bottom plate is placed in 37 DEG C of baking ovens after scribing line and dries 2 Hour, it is sealed in Fresco Bag, built-in desiccant, validity period is 15 months.
The firing of 2.3 colloidal golds
Colloidal gold is fired according to reduction of sodium citrate method: all glasswares that is used to fire, save colloidal gold are placed on sour cylinder Middle immersion for 24 hours, is rinsed well after taking-up and is dried for standby.100mL deionized water and 1mL1% gold chloride are taken, is heated to boiling.Dropwise 1% trisodium citrate of 1.5mL is added, until colour stable is at red.Hereafter continue to boil 15min, be cooled to room temperature.
The preparation of 2.4 colloidal gold probes
The colloidal gold solution 0.2%K that will be prepared2CO3PH to 5.0 is adjusted, recombinant protein rSPG is added dropwise, is shaken 15min stands 15min.10mL10%PEG20000 stabilizing solution is added, room temperature shakes 15min, stands 15min.Solution carries out 2000rpm is centrifuged 20min, takes supernatant to carry out 10000rpm centrifugation 30min again, takes precipitating, precipitating is resuspended with 20mLTBS.By this For colloidal gold probe uniform fold in gold-labelled pad, -20 DEG C of freezings 2h, -80 DEG C of freezing 2h are placed on vacuum in multi-functional freeze dryer Freeze-drying.
2.5 the preparation of colloidal gold immuno-chromatography test paper strip
Test strips are prepared as shown in Figure 6, and recombination VP6 albumen is diluted to 1.5mg/mL, 0.75mg/mL and 0.375mg/mL tri- Kind concentration.NC film is affixed on bottom plate, and to recombinate VP6 albumen as detection line (T line), albumen rSPG is nature controlling line (C line), in colloid Point sample on golden spotting system, scribing line amount are 1 μ L/cm.Scribed NC film bottom plate is placed in 37 DEG C of baking ovens, dries overnight.Successively Gold-labelled pad, sample pad, blotting paper are affixed on bottom plate, with gold mark cutting machine slitting, width 3mm.Test strips are put into sealing test paper In cylinder, and it is added 4 DEG C of desiccant and is sealed.
2.6 test strips criterion establish porcine rotavirus antibody test test strips using colloidal gold immunochromatographimethod skill Art, after test strips are inserted into positive sample, the r-SPG albumen with His label of gold label on the IgG and gold-labelled pad in sample In conjunction with compound is formed, compound is during coated film migrates, the specific IgG and VP6 on detection line (T) in sample Gold label His- r-SPG albumen-IgG- antigenic compound is formed, is developed the color at detection line, the gold then having more than needed at nature controlling line It marks the anti-His tag monoclonal antibody of mouse at His-r-SPG albumen-IgG compound and nature controlling line to form the anti-His of mouse to mark Clonal antibody-gold of signing a bill marks His-r-SPG albumen-IgG compound, to make nature controlling line (C) to develop the color, conversely, negative sample When, detection line (T) is nonantigenic, cannot form gold label His- r-SPG albumen-IgG- antigenic compound, detection line (T) It does not just develop the color, it is anti-that the anti-His tag monoclonal of mouse can only be formed in conjunction with the anti-His tag monoclonal antibody of the mouse on nature controlling line Body-gold marks His-r-SPG albumen composition and develops the color.So we have formulated the result judgement standard of test strips are as follows: sun Property detection line at (T) and nature controlling line (C) respectively there is an aubergine band, be determined as the positive；Feminine gender is only at nature controlling line (C) there is an aubergine band, do not occur an aubergine band at detection line (T), be determined as feminine gender.In vain in Quality Control (C) occurs without obvious band at line, and it is invalid to be determined as.
The result of 2.7 colloidal gold strips detects
Take porcine rotavirus mouse positive serum with the dilution of 1:10,1:20,1:50,1:100.The positive that will have been diluted Serum is dripped respectively in sample pad, stands observation.If T, C line develops the color, result is the positive；If only C line develops the color, result For feminine gender；If only T line develops the color, test strips are invalid.
(table 1) as the result is shown, the test strips can react with porcine rotavirus positive serum.Wherein protein content is 1.5mg/mL is swift in response and obviously, and protein content is that 0.75mg/mL has reaction but unobvious recombinant protein amount is 0.375mg/mL Test strips reaction it is then unobvious, therefore judge that the scribing line optium concentration of recombinant protein is about 1.5mg/mL；According to various concentration Serum testing result, can obtain serum optimum dilution degree is 1:10.
Recombinant protein and the positive serum reaction result of different extension rates of 1 various concentration of table label
2.8 test strips sensibility.Using 3 batches of porcine rotavirus antibody test test strips of trial-production to known negative sample, morbidity Animal blood serum sample is detected, the results show that 10 parts of known negative blood serum samples of detection, result is feminine gender；To 20 parts Know that affected animal blood serum sample recall rate is 100%(20/20).
<110>China Agriculture Academe Shanghai Veterinary Institute (China Animal Health and Epidemiology Center, branch center, Shanghai), Buddhist nun irrigates Northey (Suzhou) bioengineering Co., Ltd
<120>a kind of 6 gene of the rotavirus vp that amino acid sequence morphs and immuno-chromatographic test paper strip
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 444
<212> DNA
<213>VP6 full length protein sequence
<400>GGATCCAAAGATGCCCGCGACAAAATCGGCGGCAACGATTTTCAGACCGGCGGTATTGGTGGTAGCG TGGAAAATGCCCGTGGTGGTGCACGTGAAAGCCAGCGCAATGGTATTGCCCCGCAGAGTGAAGGCAGCTTCGACAAC AGCAGTGACTACATCGAGAACTGGAACCTGCAGAATCGCCGCCAGGGTGGTAATCGTAGTCAGCCGGCCCATGATAA TGGCAGCAATGCCCCGGCCAATATTGGTGGCCGTTTTAGCTTTCCGCGCGTGATCAATAGCGCCGATGGTGCAACCA CCGGTAGCCCGAATATGACCCCGGCCGTGGCCAATCTGTTTCCGCAGGGTGGTGCAATTCCGGTTGGTCCGGTGTTT CCGCCGGGCATGAATTGGACCGAACTGATCACCAACTATTCTCCGAGCCGTGAAGATAATTAAAAGCTT
<210> 2
<211> 145
<212> DNA
<213>VP6 protein sequence
<400>GSKDARDKIGGNDFQTGGIGGSVENARGGARESQRNGIAPQSEGSFDNSSDYIENWNLQNRRQGGNR SQPAHDNGSNAPANIGGRFSFPRVINSADGATTGSPNMTPAVANLFPQGGAIPVGPVFPPGMNWTELITNYSPSRED N

Claims (5)

1. a kind of immuno-chromatographic test paper strip is equipped with carrier board, sample pad, gold conjugation pad, laminate film, detection line, nature controlling line And absorption pad；The sample pad, gold conjugation pad, laminate film and absorption pad are successively pasted onto carrier board upper surface, sample pad One end be located on one end of gold conjugation pad, the other end of gold conjugation pad is located on one end of laminate film, absorption pad One end be located on the other end of laminate film, detection line, nature controlling line are sequentially arranged on laminate film；VP6 weight is coated at detection line Histone, coating selects streptococcus recombinant protein (rSPG) at nature controlling line.
2. a kind of immuno-chromatographic test paper strip as described in claim 1, the amino acid sequence such as SEQ of VP6 recombinant protein therein Shown in ID NO.2.
3. a kind of immuno-chromatographic test paper strip as claimed in claim 2, the wherein nucleotide sequence of VP6 recombinant protein such as SEQ ID Shown in NO.1.
4. a kind of preparation method of colloidal gold immuno-chromatography test paper strip as described in claim 1, which comprises
1) protein expression: preparation expression, the expression albumen for purifying a kind of rotavirus vp that amino acid sequence morphs 6；
2) point sample of nitrocellulose filter: being diluted to 0.5mg/ml for VP6, is used for the scribing line of detection line (T line), mouse is resisted His tag monoclonal antibody is diluted to scribing line of the 0.5mg/ml for nature controlling line (C line)；NC film is affixed on bottom plate, Zhi Houyong Colloidal gold spotting system is crossed, and scribing line amount is 1 μ l/cm, and it is small that bottom plate is placed in 37 DEG C of baking ovens drying 2 after scribing line When, it is sealed in Fresco Bag, built-in desiccant, validity period is 15 months；
3) firing of colloidal gold: colloidal gold is fired according to reduction of sodium citrate method: by all glass that is used to fire, save colloidal gold Glass vessel, which are placed in sour cylinder, to be impregnated for 24 hours, is rinsed well and is dried for standby after taking-up；100mL deionized water and 1mL1% gold chloride are taken, It is heated to boiling；1% trisodium citrate of 1.5mL is added dropwise, until colour stable is at red；Hereafter continue to boil 15min, it is cold But to room temperature；
4) preparation of colloidal gold probe: the colloidal gold solution 0.2%K that will be prepared2CO3PH to 5.0 is adjusted, recombination is added dropwise Albumen rSPG shakes 15min, stands 15min；10mL10%PEG20000 stabilizing solution is added, room temperature shakes 15min, stands 15min；Solution carries out 2000rpm and is centrifuged 20min, takes supernatant to carry out 10000rpm centrifugation 30min again, takes precipitating, precipitating is used 20mLTBS is resuspended；By this colloidal gold probe uniform fold in gold-labelled pad, -20 DEG C of freezings 2h, -80 DEG C of freezing 2h are placed on more Vacuum freeze-drying in function freeze dryer；
5) preparation of colloidal gold immuno-chromatography test paper strip；Will recombination VP6 albumen be diluted to 1.5mg/mL, 0.75mg/mL and Tri- kinds of concentration of 0.375mg/mL；NC film is affixed on bottom plate, and to recombinate VP6 albumen as detection line (T line), albumen rSPG is nature controlling line (C line), the point sample on colloidal gold spotting system, scribing line amount are 1 μ L/cm；Scribed NC film bottom plate is placed in 37 DEG C of baking ovens, overnight Drying；Successively gold-labelled pad, sample pad, blotting paper are affixed on bottom plate, with gold mark cutting machine slitting, width 3mm；Test strips are put Enter to seal in test paper cylinder, and is added 4 DEG C of desiccant and is sealed.
5. the rotavirus colloidal gold immuno-chromatography test paper strip of claim 1 is examined in preparation diagnosis, prevention or treatment rotavirus Application in disconnected reagent or drug.
CN201910844679.5A 2019-09-07 2019-09-07 A kind of 6 gene of the rotavirus vp that amino acid sequence morphs and immuno-chromatographic test paper strip Pending CN110470829A (en)

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