CN101508999A - Recombinant toxoplasma protein and uses thereof - Google Patents
Recombinant toxoplasma protein and uses thereof Download PDFInfo
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- CN101508999A CN101508999A CNA2009100801352A CN200910080135A CN101508999A CN 101508999 A CN101508999 A CN 101508999A CN A2009100801352 A CNA2009100801352 A CN A2009100801352A CN 200910080135 A CN200910080135 A CN 200910080135A CN 101508999 A CN101508999 A CN 101508999A
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Abstract
The invention relates to a Toxoplasma gondii protein for clinical diagnosis of Toxoplasma gondii infection, and a nucleotide sequence encoding the protein. The protein comprises plasmids of the nucleotide sequence and procaryotic host cells. The invention also comprises a method for preparing the protein and application thereof.
Description
Technical field
The present invention relates to fields such as genetically engineered, vaccine development and diagnostic reagent, relate to a kind of recombinant toxoplasma protein and application thereof particularly.
Background technology
Toxoplasma gondii (Toxoplasma gondii Nicolle ﹠amp; Manceaux, 1908) be the enteron aisle coccidia of feline, find that by French scholar Nicolle and Manceaux it is arc that polypide is, the called after toxoplasma gondii in just combing toe mouse (Ctenodactylus gondii) monocyte.This worm is worldwide distribution, and people and many animals can both infect, and cause the toxoplasmosis of infecting both domestic animals and human, especially when host immune function is low, can cause serious consequence, belongs to opportunistic diseases protozoon (opportunistic protozoan).
Congenital toxoplasmosis only betides unigravida woman, propagates through placental blood flow.Be contaminted fetus or baby's majority shows as inapparent infection, and symptom just appears in several months even several years after the birth that has; Also can cause pregnant woman's miscarriage, premature labor, monster or stillbirth, the You Yizao pregnancy period infects, monster incidence height.Show that according to the study occurring symptom during baby due or lopsided person's case fatality rate takes place is 12%, and 80% spiritedness dysplasia in the survival, 50% has visual disorder
[1]Obstacle with hydrocephalus, cerebral calcification kitchen range, retinochoroiditis and spirit, motion is a congenital toxoplasmosis typical case symptom.
Acquired toxoplasmosis can present different clinical manifestations with reactivity of organism because of polypide attacks the position.Most inapparent infection persons, when suffering from malignant tumour, implement organ transplantation, accept under the iatrogenic immunocompromised host situations such as immunosuppressor, radiotherapy, cytotoxic agent for a long time or congenital, acquired immunity handicapped, all can make the inapparent infection state transfer acute severe to as AIDS patient, pregnant women etc., make original condition worse.Quick, the accurate detection method of setting up the TOX infection is the key point that prevention TOX infects.
The arch insect infection diagnostic method mainly contains immunology detection, directly microscopy, pathogen isolation and polymerase chain reaction (PCR) detection etc. at present
[1-4]Immunological method is owing to simple and efficient to handlely be suitable for clinical use.The invention provides a kind of recombinant antigen of producing with gene engineering method, can avoid that low or poor specificity causes false positive to reduce problems such as detection specificity owing to proteantigen purity, for the detection of arch insect infection provides more special, raw material accurately.
In the world, lack at present under the situation of effectively treating the arch insect infection medicine, become a kind of important medical intervention means by vaccination prevention arch insect infection.Simultaneously, the development of toxoplasma gondii vaccine also is to carry out one of key areas of toxoplasma gondii research.Toxoplasma gondii recombinant protein provided by the invention has high specific, strong immunogenicity and the complete antigenic determinant of trying one's best, and has actual application value in toxoplasma gondii vaccine development field.
Summary of the invention
The purpose of this invention is to provide a kind of recombinant toxoplasma protein, another object of the present invention provides the application of this recombinant toxoplasma protein in the preparation detection kit.
Technical scheme of the present invention
The invention provides a kind of recombinant nucleic acid of the toxoplasma protein of encoding, its nucleotide sequence is shown in sequence 1.Toxoplasma protein by this recombinant DNA sequence coding is provided simultaneously, and its aminoacid sequence is as sequence: shown in 2.
The present invention also provides a kind of expression vector pET-28a-TOX of toxoplasma protein, and it is that described nucleotide sequence shown in the sequence 1 is inserted into the recombinant plasmid that obtains on the plasmid pET-28a, and its plasmid map as shown in Figure 2.Expression vector pET-28a-TOX is imported in the intestinal bacteria, obtain expressing the engineering strain of toxoplasma protein.
The present invention utilizes nucleotide sequence shown in the sequence 1 to prepare toxoplasma protein by gene engineering method and can realize as follows:
1) obtains to have the nucleotide sequence shown in the sequence 1;
2) this nucleotide sequence is imported plasmid, preferred pET-28a plasmid;
3) this plasmid is imported prokaryotic host cell, preferred e. coli host cell is more preferably in BL21 (DE3) bacterial strain;
4) under the condition that helps described nucleotide sequence expression, cultivate described host cell;
5) recombinant protein that recovery, purifying and renaturation are expressed.
The invention still further relates to the composition and the test kit that comprise toxoplasma protein of the present invention.Described composition can be used as the reagent that detects arch insect infection.Described composition can be used as the test kit that detects arch insect infection.
Toxoplasma protein antigen provided by the invention has high specific, strong immunogenicity and the complete antigenic determinant of trying one's best, therefore, it may occur to persons skilled in the art that, toxoplasma protein of the present invention can be used for preparing the toxoplasma gondii vaccine, as subunit vaccine etc., equally, the nucleotide sequence shown in sequence 1 disclosed in this invention also can be used to prepare dna vaccination.Therefore the invention still further relates to the vaccine of the toxoplasma gondii that comprises toxoplasma protein of the present invention or nucleic acid.
The detailed description of invention
The invention discloses a kind of nucleotide sequence shown in sequence 1 of the toxoplasma protein of encoding, utilize this nucleotide sequence to prepare the method for toxoplasma protein, the toxoplasma protein that comprises aminoacid sequence shown in the sequence 2 by this method preparation, and comprise this proteic composition and test kit, they are also disclosed simultaneously in the application that detects arch insect infection.In addition, toxoplasma protein of the present invention and nucleic acid of the present invention also can be used for the preparation of toxoplasma gondii vaccine.
One embodiment of the invention relate to a kind of nucleotide sequence shown in sequence 1 of the toxoplasma protein of encoding.Base be numbered conventional 5 ' to 3 ' in proper order.
Nucleotide sequence shown in the sequence 1 can be by the method preparation of this area routine, preferably according to the segmental dna sequence dna of purpose, P24, the P29, P30 and the P35 that are TOX go up the cDNA sequence of purpose peptide section and the restriction enzyme site on the plasmid, design four couples of PCR primer (P1, P2), (P3, P4), (P5, P6) and (P7, P8).P1 and P2 the 259th to the 429th Nucleotide of P24 that is used to increase, P3 and P4 the 88th to the 345th Nucleotide of P29 that is used to increase, P5 and P6 the 652nd to the 816th Nucleotide of P30 that is used to increase, P7 and P8 the 310th to the 663rd Nucleotide of P35 that is used to increase; Primer P1 and P8 have the restriction enzyme site of NdeI and XhoI respectively, primer P2 and P3 order are complementary, and jointly corresponding to P24 above-mentioned segmental 3 ' the terminal and above-mentioned segmental 5 ' end sequence of P29, primer P4 and P5 order are complementary, and jointly corresponding to P29 above-mentioned segmental 3 ' the terminal and above-mentioned segmental 5 ' end sequence of P30, primer P6 and P7 order are complementary, and jointly corresponding to P30 above-mentioned segmental 3 ' the terminal and above-mentioned segmental 5 ' end sequence of P35.
The present invention can improve the expression productive rate when nucleic acid molecule of above-mentioned nucleotide sequence is adopted suitable carrier and host cell expression greatly.
One embodiment of the invention relate to the method that the nucleotide sequence of application shown in sequence 1 prepares toxoplasma protein.According to conventional methods, the nucleic acid molecule of nucleotide sequence shown in sequence 1 that contains the toxoplasma protein of encoding can be connected in the expression vector, then by the ordinary method transformant.Usually, preferred prokaryotic organism are used for the initial clone of dna sequence dna and are used for vector construction of the present invention.For example, intestines section bacillus such as intestinal bacteria.
The heterozygosis plasmid that code book is invented proteic nucleic acid and pET-28a formation has the stability of height, helps proteic expression of the present invention.
In one embodiment of the invention, as shown in Figure 2, preparation contains the expression construct of the nucleic acid molecule and the pET-28a plasmid of nucleotide sequence shown in the sequence 1, and this construct is transformed BL21 (DE3), after 4～5 hours, collect thalline through the IPTG inducing culture.Can adopt the conventional resulting albumen of purification process purifying.
One embodiment of the invention relate to the toxoplasma protein with method for preparing, purifying, and this albumen has aminoacid sequence shown in the sequence 2.
One embodiment of the invention relate to composition and the test kit that comprises toxoplasma protein of the present invention.Described composition or test kit can be prepared into reagent or the kit form that detects the toxoplasma gondii arch insect infection.
Another embodiment of the present invention relates to the arch insect infection monitoring reagent box that contains toxoplasma protein of the present invention, is used for clinically easily and fast and arch insect infection accurately.
" test kit " described herein is meant and utilizes albumen of the present invention to finish that arch insect infection detects and reagent set that assembly is made.This test kit is used to diagnose arch insect infection.Test kit of the present invention can comprise other a plurality of containers, wherein can contain to detect used standard substance antibody, the enzyme of antibody or process mark, substrate or damping fluid etc. respectively.In this test kit, comprise that also label and package insert are in order to provide the operation instruction of test kit.Also can comprise other material that meets user's needs, as microtiter plate etc.
Be used for the test kit that arch insect infection detects, toxoplasma protein of the present invention also can be through mark.Specifically can use marks such as enzyme, metallo-chelate.Preferred mark enzyme for example, horseradish peroxidase, peroxidase, alkaline phosphatase etc.Preferred metallics has Radioactive colloidal gold etc.
With above-mentioned marker bonded method be known.When marker was enzyme, its substrate and developer can be used for measuring its activity.When using horseradish peroxidase, with 3 ', 3 ', 5 ', 5 ' ,-tetramethyl benzidine is as substrate solution, and uses the TMB Color Appearance System.When using peroxidase, with H
2O
2As substrate solution, and with the amino antipyrine of O-Phenylene Diamine, 4-etc. as developer.When using alkaline phosphatase, available ortho-nitrophenyl phosphoric acid, p-nitrophenyl phosphoric acid etc. are as substrate.
The present invention relates to the arch insect infection detection method, described detection comprises the reagent or the test kit of toxoplasma protein of the present invention by application, adopt conventional serological method to realize, for example, can use the reagent that comprises toxoplasma protein of the present invention or test kit to detect resisting toxoplasmosis antibody in the body fluid such as serum.The method that detects resisting toxoplasmosis antibody has indirect test, (solid phase can be a solid phase commonly used well-known to those skilled in the art as toxoplasma protein antigen of the present invention being adsorbed in solid phase carrier, for example polystyrene, immunochromatography are with filter paper etc.), add serum to be checked then, exist if any corresponding antibodies, then form antigen-antibody complex with antigen on carrier, the washing back adds enzyme mark anti-antibody or colloid gold label anti-antibody, the colour developing observations.Described indirect test method is ELISA method, spot gold-marking immunity percolation process etc. for example.
Any biological sample as long as they contain toxoplasma antibody, with regard to available albumen of the present invention, comprises this proteic composition or test kit and detects.
Used all reagent or the test kit that comprises toxoplasma protein of the present invention all comprises within the scope of the invention in above-mentioned arch insect infection detects.
(3) beneficial effect
Adopt the diagnostic kit of recombinant toxoplasma protein preparation provided by the invention, compare, have advantages such as high specificity, sensitivity height, well satisfied the needs of arch insect infection clinical diagnosis with the similar test kit on the market.
Description of drawings
The gel electrophoresis figure of Fig. 1 pcr amplification product;
Fig. 2 is the structure schema of expression plasmid pET-28a-HSV;
Fig. 3 induces back thalline 12%SDS-PAGE electrophorogram, and wherein M represents Marker, 1 and 2 expression target proteins.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting protection scope of the present invention.
The preparation of embodiment 1 recombinant toxoplasma protein
1.1 toxoplasma protein epitope screening and goal gene clone
Whole aminoacid sequences by the Computer Analysis toxoplasma gondii filter out proteic dominant antigen epi-position in the arc polypide, comprise 57 amino acid of P24 from terminal the 87th to the 143rd of N-, P29 is from 86 amino acid of terminal the 30th to the 115th of N-, 118 amino acid of P30 from 55 amino acid of terminal the 218th to 272 of N-and P35 from terminal the 104th to 221 of N-.The dna sequence dna of described recombinant protein is shown in SEQ ID No.1, wherein, P24 purpose peptide segment DNA sequence is the 259th to the 429th Nucleotide of P24, P29 purpose peptide segment DNA sequence is the 88th to the 345th Nucleotide of P29, P30 purpose peptide segment DNA sequence is the 652nd to the 816th Nucleotide of P30, and P35 purpose peptide segment DNA sequence is the 310th to the 663rd Nucleotide of P35.
According to the segmental dna sequence dna of purpose, promptly the P24 of TOX, P29, P30 and P35 go up the cDNA sequence of purpose peptide section and the restriction enzyme site on the plasmid, design four pairs of PCR primers (P1, P2), (P3, P4), (P5, P6) with (P7, P8).P1 and P2 the 259th to the 429th Nucleotide of P24 that is used to increase, P3 and P4 the 88th to the 345th Nucleotide of P29 that is used to increase, P5 and P6 the 652nd to the 816th Nucleotide of P30 that is used to increase, P7 and P8 the 310th to the 663rd Nucleotide of P35 that is used to increase; Primer P1 and P8 have the restriction enzyme site of NdeI and XhoI respectively, primer P2 and P3 order are complementary, and jointly corresponding to P24 above-mentioned segmental 3 ' the terminal and above-mentioned segmental 5 ' end sequence of P29, primer P4 and P5 order are complementary, and jointly corresponding to P29 above-mentioned segmental 3 ' the terminal and above-mentioned segmental 5 ' end sequence of P30, primer P6 and P7 order are complementary, and jointly corresponding to P30 above-mentioned segmental 3 ' the terminal and above-mentioned segmental 5 ' end sequence of P35.
With toxoplasma gondii culture (international standard strain RH strain Inst. of Viruses, China Preventive Medicine Science Academy gives) is template, with primer P1 and P2 amplification P24 target DNA fragment, with primer P3 and P4 amplification P29 target DNA fragment, with primer P5 and P6 amplification P30 target DNA fragment, with primer P7 and P8 amplification P35 target DNA fragment.The P24 target DNA fragment and the P29 target DNA fragment that obtain with first round pcr amplification are template, add primer P1 and P4, amplification P24P29 target DNA fragment.The P30 target DNA fragment and the P35 target DNA fragment that obtain with first round pcr amplification are template, add primer P5 and P8, amplification P30P35 target DNA fragment.Taking turns P24P29 and the P30P35 target DNA fragment that pcr amplification obtains with second is template, adds primer P1 and P8, amplification P24P29P30P35 target DNA fragment.Obtain complete target DNA recombinant fragment: P24P29P30P35.Amplified production detects through agarose gel electrophoresis, and the result as shown in Figure 1.Cutting contains the blob of viscose of target DNA band, and (lead to-Beijing TAKARA company name of product available from the six directions: TAKARA MiniBEST Plasmidpurification) reclaim target DNA, operation is undertaken by product description with DNA fast purifying test kit.
1.2 the structure of expression vector pET-28a-TOX and evaluation
PET-28a carrier (available from magnificent Bioisystech Co., Ltd) and pcr amplification product P24P29P30P35 target DNA fragment are through NdeI and XhoI double digestion, product purification (adopt TAKARA MiniBEST Plasmid purification test kit, lead to-Beijing TAKARA company available from the six directions) is after T
4Dna ligase is connected with carrier, connect product and be transformed into e. coli bl21 (DE3) competence (available from magnificent Bioisystech Co., Ltd), coat the LB that contains kantlex and be inverted overnight incubation for dull and stereotyped last 37 ℃, select the dull and stereotyped bacterium colony of going up growth next day, alkaline lysis extracting plasmid, agarose gel electrophoresis selects suspicious recombinant plasmid to carry out pcr amplification as template, recon plasmid that amplified production is arranged through restriction endonuclease NdeI and EcoRI double digestion, evaluation, wherein there are 3 positive recons of recon.Select a positive recombinant to send match hundred victory companies order-checking to identify from 3 positive recombinants, the result shows and has inserted goal gene on this plasmid really, and direction of insertion is correct, with this recombinant plasmid called after expression plasmid pET-28a-TOX.
1.3 the structure of the engineering bacteria of express recombinant toxoplasma protein
Change expression plasmid pET-28a-TOX over to expression strain BL21 (DE3) (available from magnificent Bioisystech Co., Ltd), coat the LB that contains kantlex and be inverted overnight incubation for dull and stereotyped last 37 ℃, select the dull and stereotyped bacterium colony LB culture medium culturing that contains kantlex that goes up growth next day, with IPTG abduction delivering 5 hours, induce thalline to analyze ((U.S.) J. Sa nurse Brooker (JosephSambrook), (U.S.) D.W. Russell (David W.Russell) work with 12%SDS-PAGE
[5], Huang Peitang etc. translate. and the molecular cloning experiment guide. Beijing: Science Press, 2002.), the result determines that the bacterial strain of expressing toxoplasma protein is required engineering strain, as shown in Figure 3 with the freezing preservation of glycerine.
1.4 the preparation of recombinant toxoplasma protein and purifying
The engineering strain of the expression toxoplasma protein that inducing culture has made up, with IPTG abduction delivering 5 hours, centrifugal collection thalline, add cellular lysate liquid (50mm pH8.0Tris-Cl with 1:10 (W/V), 50mm NaCl, 50% glycerine), adding the magnetic agitation rotor stirred 30 minutes, through carrying out ultrasonic bacteria breaking (ice bath, power 200W, ultrasonic 3 seconds, 5 seconds at interval, ultrasonic 80 times), (Glutathione Sepharose 4B post material 50ml adorns post to the Histidine affinity column, and 400mlPBS damping fluid balance is crossed post, flow velocity 5ml/min; Ultrasonic supernatant liquor after centrifugal is gone up sample, flow velocity 3ml/min with 200mlPBS dilution back; The 400mlPBS damping fluid is washed post, flow velocity 5ml/min; 250ml elution buffer wash-out, flow velocity 3ml/min, Ultraviolet Detector 280nm detect, and collect elution peak) and ion-exchange chromatography (100mlChelating Sepharose FF adorns post, the NiCl of 300ml200mm
2Cross post, flow velocity 5ml/min; The 500ml level pad is washed notes, flow velocity 10ml/min; Histidine affinity column purifying is collected the liquid upper prop, flow velocity 3ml/min, the 500ml level pad is washed notes, flow velocity 5ml/min; With the elution buffer wash-out that contains imidazoles 20mm, 50mm, 100mm, each 100ml of 200mm respectively, Ultraviolet Detector 280nm detects absorption value, collects elution peak; SDS-PAGE electrophoresis detection purified components) obtains the recombinant protein of purifying.
1.5 recombinant protein Western-blot checking
For verifying that reorganization TOX protein and anti-TOX antiserum(antisera) are reactive and the goal gene of recombinating obtains expression and has antigenicity, test with the Western-blot method.Positive serum is: 10 parts of rabbit anti-TOX antibody positive serum and the 30 parts of anti-TOX antibody positive of people serum (detecting confirmation through the ELISA method) with TOX culture immune rabbit results.Negative serum is: 10 parts contrast normal rabbit serum and the 30 parts of anti-TOX negative antibody of people serum (detecting confirmation through the ELISA method).Result such as table 1.
Table 1 recombinant protein Western-blot verifies the result
| Serum sample | Total routine number | Positive routine number | Negative routine number | Positive rate | Negative recall rate |
| The anti-TOX antibody positive of rabbit serum | 10 | 10 | 0 | 100％ | 0 |
| The contrast normal rabbit serum | 10 | 0 | 10 | 0 | 100％ |
| The anti-TOX antibody positive of people serum | 30 | 30 | 0 | 100％ | 0 |
| The anti-TOX negative antibody of people serum | 30 | 0 | 30 | 0 | 100％ |
The result shows, 10 parts of rabbit anti-TOX antibody positive serum and the 30 parts of anti-TOX antibody positive of people serum with TOX culture immune rabbit gained all produce positive reaction with antigen expressed, and 10 parts of contrast normal rabbit serums and 30 parts of people's anti-TOX negative antibody serum and antigen expressed all produce negative reaction.Results suggest reorganization goal gene obtains to express, and reorganization TOX protein has tangible cross reactivity with full TOX protein, and has very strong specificity, has the practical application meaning.
The preparation of embodiment 2 toxoplasmosis IgM antigen testing reagent boxes and performance calibrating
2.1 the preparation of toxoplasmosis IgM antigen testing reagent box
The reorganization TOX albumen that embodiment 1 is made is used as the test kit labelled antigen, measures TOX IgM antibody with colloidal gold method.
The development of this test kit and use as follows:
1) test kit principle: this product adopts immunocapture method principle, and with anti-people IgM (anti-people μ chain) antibody sandwich nitrocellulose filter, colloid gold label genetically engineered recombinant toxoplasma (TOX) antigen is tracer.Add serum to be checked during use, as containing anti-TOX specific IgM antibodies in the sample, then can combine with the anti-human IgM antibody on film surface and form mixture, this mixture combines with the TOX antigen of colloid gold label and presents the red-purple band.
2) test kit performance optimization
(collect how tame hospital with positive and negative quality control product through the resisting toxoplasmosis IgM of clinical verification antibody positive, negative serum, recheck screening with the resisting toxoplasmosis IgM antibody ELISA test kit that Italian SORIN company provides, the resisting toxoplasmosis IgM positive that obtains, negative serum is established as the positive and negative quality control product of enterprises) be specimen, adopt the square formation titration to determine the working concentration of best anti-people IgM (anti-people μ chain) and TOX antigen colloidal gold (TOX-Ag.G) binding substances, result such as table 2, drawn by the result, anti-people IgM (anti-people μ chain) antibody line bag is 2.0mg/ml by concentration, the TOX-Ag.G mixture is carried gold in concentrated stoste to bag between diluting a times and is filled up.
The selection of best anti-people IgM of table 2 (anti-people μ chain) and TOX-Ag.G binding substances working concentration
-: negative reaction; ±: probable positive, nature controlling line mays be seen indistinctly; +: nature controlling line appears in positive reaction;
++: than strong positive reaction, nature controlling line is clear; +++strong positive reaction, nature controlling line is clear thick.
Determining that anti-people IgM (anti-people μ chain) antibody line bag is that 2.0mg/ml, TOX-Ag.G mixture are carried on the golden basis of filling up to bag between diluting a times in concentrated stoste by concentration, with the inner Quality Control positive and negative quality control product (source and standard are the same) is specimen, and it is 1.0 μ l/cm that best anti-people IgM (anti-people μ chain) antibody line amount (the metal spraying amount is 20 μ l/cm) is selected in titration.The results are shown in Table 3.
Determining of the best anti-people IgM of table 3 (anti-μ chain) antibody line amount
-: negative reaction; ±: probable positive, nature controlling line mays be seen indistinctly; +: nature controlling line appears in positive reaction;
++: than strong positive reaction, nature controlling line is clear; +++strong positive reaction, nature controlling line is clear thick.
Determining that anti-people IgM (anti-people μ chain) line concentration is 2.0mg/ml, the line amount is that 1.0 μ l/cm and toxoplasma antigen colloidal gold composite specking concentration are to concentrate stoste to the basis between diluting a times, with the inner Quality Control positive and negative quality control product (source and standard are the same) is specimen, and adopting the square formation titration to select best Radioactive colloidal gold binding substances specking amount is 25.0 μ l/cm.The results are shown in Table 4.
Determining of the best antigen colloidal gold mixture of table 4 specking amount
-: negative reaction; ±: probable positive, nature controlling line mays be seen indistinctly; +: nature controlling line appears in positive reaction; ++: than strong positive reaction, nature controlling line is clear; +++strong positive reaction, nature controlling line is clear thick.
On the basis of determining antigen colloidal gold mixture package amount, select the bag of nature controlling line rabbit resisting toxoplasmosis antibody by concentration.Rabbit resisting toxoplasmosis antibody sandwich amount is 1.0 μ l/cm, and bag is 5.0mg/ml by concentration.The results are shown in Table 5.
The selection of table 5 nature controlling line rabbit resisting toxoplasmosis antibody sandwich concentration
-: negative reaction; ±: probable positive, nature controlling line mays be seen indistinctly; +: nature controlling line appears in positive reaction;
++: than strong positive reaction, nature controlling line is clear; +++strong positive reaction, nature controlling line is clear thick.
Anti-people μ chain line bag by after, seal with following buffering system respectively, the comparison sealing effect, the result shows and contains PEG
20000Sensitivity of closed system sealing back and specificity all descend to some extent, other sealings and the results that do not seal are as broad as long, so select not seal nitrocellulose filter.The results are shown in Table 6.
The Sptting plate of the different closed systems of table 6 relatively
-: negative reaction; ±: probable positive, nature controlling line mays be seen indistinctly; +: nature controlling line appears in positive reaction;
++: than strong positive reaction, nature controlling line is clear; +++strong positive reaction, nature controlling line is clear thick.
3) preparation of test kit
A, NC film precut → adhesive back → bag by mouse-anti people IgM (anti-people μ chain) antibody (hundred peace world scientific ﹠ technical corporation buy by Beijing) and rabbit resisting toxoplasmosis antibody (hundred peace world scientific ﹠ technical corporation buy by Beijing) line bag quilt on the NC film → drying.
The specking bag quilt → drying → cutting of b, synthetic Radioactive colloidal gold → colloidal gold solution check → antigenic mark Radioactive colloidal gold → antigen colloidal gold mixture check → antigen colloidal gold mixture.
C, work in-process (master card) assemble → cut into chromatography strip → inspection of semifinished product → assembling test card → pack test card.
D, assembling finished product box → inspection after construction → preservation.
4) test kit working method:
A. open the packaging of aluminium foil bag of test card, take out test card.
B. test card being inclined to the application of sample nose end is lower than the other end and is no less than 1.0cm.
C. get 100 μ l serum to be checked and join in the circular sample hole, room temperature (15 ℃-30 ℃) was placed 25 minutes.
Assay is judged:
A.25 minute observe and the record result.
B. positive: two red-purple lines bands (C and T position) appear in the interpretation window; Negative: a red-purple lines band (C position) only appears in the interpretation window.
C. invalid: interpretation window C does not have red-purple lines band in the position.
2.2 test kit Performance Detection experiment
Specificity (accuracy) is measured: the Radioactive colloidal gold catch assay method by previous experiments is determined, detect several other serum materials, the observing response specificity.The result shows, uses test kit of the present invention to detect 15 parts of negative serums (clinical definite), and coincidence rate is 100%; Detect 50 parts of Rheumatoid factors, polyclonal positive serum samples, 50 parts of hepatitis C positive serum samples, 50 parts of syphilis positive serum samples etc., none positive shows that Rheumatoid factors, polyclonal etc. can not cause the false positive of test kit substantially, and specificity is fine.
Sensitivity (specificity) is measured: by the Radioactive colloidal gold catch assay method that previous experiments is determined, detect the susceptibility of this test kit.Use test kit of the present invention to detect 15 parts of positive serums (clinical definite), coincidence rate is 100%.
The performance calibrating of embodiment 3 toxoplasmosis IgM antigen testing reagent boxes (euzymelinked immunosorbent assay (ELISA))
The substrate that the reorganization TOX albumen that embodiment 1 is made prepares as the test kit enzyme-labelled antigen is measured TOX IgM antibody with the ELISA method.
3.1 this test kit principle and component are as follows:
(1) test kit principle: this strain is wrapped the microwell plate of quilt with anti-people IgM (μ chain), horseradish peroxidase (HRP) marker gene engineering reorganization TOX antigen is tracer, the TMB Color Appearance System is used the resisting toxoplasmosis IgM antibody in prize law principle detection human serum or the blood plasma.
(2) the main moiety of test kit:
1) the pre-bag by plate: be microwell plate with anti-people IgM (μ chain) antibody sandwich, 1 (9 * 12 hole);
2) enzyme conjugates: horseradish peroxidase (HRP) mark HSV2 antigen of the present invention is tracer.0.2M PB (pH7.4 ± 0.2) damping fluid 100.0ml, NaCl 8.775g, casein 1.0g, 20%Tween-20 0.25ml, normally nascent bovine serum 200.0ml, deionized water 700.0ml.1 bottle, 12ml.
3) negative control: 0.2M PB (pH7.4 ± 0.2) damping fluid 100.0ml, sucrose 50.0g, people CMVIgM negative serum 200.0ml, deionized water 700.0ml.1 bottle, 0.4ml.
4) positive control 0.2M PB (pH7.4 ± 0.2) damping fluid 100.0ml, sucrose 50.0g, people CMVIgM positive serum 0.5ml, calf serum 200.0ml, deionized water 700.0ml.1 bottle, 0.4ml.
5) concentrate washing lotion (20 *): disodium hydrogen phosphate 58.0g, sodium dihydrogen phosphate dihydrate 5.93g, sodium-chlor 175.5g, tween 20 10.0ml, its plain 0.035g of letter, deionized water adds to 1000.0ml.1 bottle, 50ml.
6) substrate solution A: Sodium acetate trihydrate 4.76g, Glacial acetic acid 0.9ml, urea peroxide 0.5g, deionized water adds to 1000.0ml.1 bottle, 6ml.
7) substrate solution B: Citric acid monohydrate Food grade 1.0g, ethylenediamine tetraacetic acid (EDTA) 0.1g, TMB 0.32g, DMF2.0ml, vitriol oil 0.1ml, methyl alcohol 50.0ml, 4%PVA 2.0ml, 4% Sulfothiorine 0.05ml, deionized water adds to 950.0ml.1 bottle, 6ml.
8) stop buffer: vitriol oil 56.25ml, deionized water 944.0ml.1 bottle, 6ml.
3.2 test kit performance calibrating
Specificity (accuracy) is measured: by catching the ELISA measuring method, detect several other serum materials, the observing response specificity.The result shows, uses test kit of the present invention to detect 15 parts of negative serums (clinical definite), and coincidence rate is 100%; Detect 50 parts of Rheumatoid factors, polyclonal positive serum samples, 50 parts of hepatitis C positive serum samples, 50 parts of syphilis positive serum samples etc., none positive shows that Rheumatoid factors, polyclonal etc. can not cause the false positive of test kit substantially, and specificity is fine.
Sensitivity (specificity) is measured: by catching the ELISA measuring method, detect the susceptibility of this test kit.Use test kit of the present invention to detect 15 parts of positive serums (clinical definite), coincidence rate is 100%.
Accuracy is measured: by catching the ELISA measuring method, detect the accuracy of this test kit.Use this test kit that positive control, the negative control of 2 parts of anti-HSV2 strong positive serum, 1 part of positive serum, 1 part of negative serum and test kit are carried out every plate diplopore, the detection of totally 10 plates, result such as table 9, the repeatability of visible test kit is good, and the CV that different serum are detected all is lower than 15%.
The accuracy of table 9 test kit
| Serum | Average OD | Maximum OD | Minimum OD | CV(％) |
| Strong positive 1 | 3.609 | 4.117 | 3.028 | 3.2 |
| Strong positive 2 | 3.208 | 3.579 | 2.436 | 4.0 |
| Positive 1 | 2.447 | 2.817 | 1.593 | 6.7 |
| Negative | 0.018 | 0.108 | 0.004 | 6.8 |
| Positive control | 2.479 | 3.193 | 2.005 | 5.3 |
| Negative control | 0.018 | 0.111 | 0.009 | 3.9 |
Sequence table
＜110〉Beijing Innote Biotechnology Co., Ltd.
＜120〉a kind of recombinant toxoplasma protein and application thereof
<160>10
<210>1
<211>948
<212>DNA
＜213〉synthetic
＜223〉nucleotide sequence of the recombinant nucleic acid of coding toxoplasma protein of the present invention
<400>1
<210>2
<211>316
<212>PRT
＜213〉synthetic
＜223〉aminoacid sequence of express recombinant toxoplasma protein of the present invention
<400>2
<210>3
<211>36
<212>DNA
＜213〉synthetic
＜223〉the present invention the 259th primer P1 of P24 that be used to increase to the 429th Nucleotide:
<400>3
<210>4
<211>33
<212>DNA
＜213〉the present invention the 259th primer P2 of P24 that be used to increase to the 429th Nucleotide:
<400>4
<210>5
<211>32
<212>DNA
＜213〉synthetic
＜223〉the present invention the 88th primer P3 of P29 that be used to increase to the 345th Nucleotide:
<400>5
<210>6
<211>35
<212>DNA
＜213〉synthetic
＜223〉the present invention the 88th primer P4 of P29 that be used to increase to the 345th Nucleotide:
<400>6
<210>7
<211>37
<212>DNA
＜213〉synthetic
＜223〉the present invention the 652nd primer P5 of P30 that be used to increase to the 816th Nucleotide:
<400>7
<210>8
<211>30
<212>DNA
＜213〉synthetic
＜223〉the present invention the 652nd primer P6 of P30 that be used to increase to the 816th Nucleotide:
<400>8
<210>9
<211>33
<212>DNA
＜213〉synthetic
＜223〉the present invention the 310th primer P7 of P35 that be used to increase to the 663rd Nucleotide:
<400>9
<210>10
<211>28
<212>DNA
＜213〉synthetic
＜223〉the present invention the 310th primer P8 of P35 that be used to increase to the 663rd Nucleotide:
<400>10
Claims (10)
1. a kind of nucleic acid of the toxoplasma protein of encoding shown in the sequence 1.
2. the carrier that comprises the described nucleic acid of claim 1.
3. the described carrier of claim 2, it is pET-28a-TOX.
4. the proteic preparation method of recombinant human herpes simplex virus is characterized in that application rights requires 1 nucleic acid construct expression vector, and express the toxoplasma protein of the described nucleic acid encoding of claim 1 in the prokaryotic host cell that this carrier was fit to.
5. the described method of claim 4 is characterized in that described expression vector is the prokaryotic expression carrier.
6. the described method of claim 5 is characterized in that described expression vector is pET-28a-TOX.
7. the arch insect infection diagnostic kit that comprises the nucleic acid of claim 1.
8. the toxoplasma gondii virus vaccine that comprises the nucleic acid of claim 1.
9. the described nucleic acid of claim 1 is in the application of preparation in preparation arch insect infection detection reagent.
10. the application of the described nucleic acid of claim 1 in preparation toxoplasma gondii vaccine.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102492696A (en) * | 2011-12-13 | 2012-06-13 | 北京英诺特生物技术有限公司 | Recombinant toxoplasma protein and application thereof |
| CN102925462A (en) * | 2012-10-29 | 2013-02-13 | 英诺特（唐山）生物技术有限公司 | Chlamydia trachomatis recombinant protein and preparation method thereof |
| CN103267846A (en) * | 2013-06-09 | 2013-08-28 | 湖南农业大学 | An ELISA kit for detecting type 2 porcine circovirus IgM antibody |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102492696A (en) * | 2011-12-13 | 2012-06-13 | 北京英诺特生物技术有限公司 | Recombinant toxoplasma protein and application thereof |
| CN102492696B (en) * | 2011-12-13 | 2014-01-29 | 北京英诺特生物技术有限公司 | Recombinant toxoplasma protein and application thereof |
| CN102925462A (en) * | 2012-10-29 | 2013-02-13 | 英诺特（唐山）生物技术有限公司 | Chlamydia trachomatis recombinant protein and preparation method thereof |
| CN103267846A (en) * | 2013-06-09 | 2013-08-28 | 湖南农业大学 | An ELISA kit for detecting type 2 porcine circovirus IgM antibody |
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