CN102533795B - Recombinant human cytomegalovirus protein and applications thereof - Google Patents
Recombinant human cytomegalovirus protein and applications thereof Download PDFInfo
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Abstract
The invention provides a human cytomegalovirus protein and also provides applications of the human cytomegalovirus protein in preparing a detection kit. Compared with similar kits in the market, a diagnostic kit prepared by adopting the coding human cytomegalovirus protein provided by the invention has the advantages of strong specificity, high sensitivity and the like, thereby, the requirement of the clinical diagnosis of human cytomegalovirus protein infection is well met.
Description
Technical field
The present invention relates to the fields such as genetically engineered, vaccine development and diagnostic reagent, relate to particularly a kind of human cytomegalic inclusion disease virus albumen and application thereof.
Background technology
Human cytomegalic inclusion disease virus (Human Cytomegalovirus is called for short HCMV) belongs to simplexvirus β subfamily, is a kind of DNA virus, and it infects in the crowd and extensively exists, and in population of China, HCMV infects quite serious.It is to cause that fetal in utero infects and grow damaged major reason that the women is subjected to the HCMV primary infection at the gestation initial stage, in the fetus an d neonate of pregnancy period primary infection HCMV, 80% can cause mental retardation, deformity and death [Middeldorp JM.Jongsma J, Haar AT, et al.Detection of immunoglobulin M and G antibodies against cytomegalovirus early and late antigend by enzyme-linked immunosorbent assay.J Clin.Microbiol, 1984; 20 (4): 763-771.].HCMV is also one of organ transplantation failure and AIDS patients accompanying infection main causes of death.Nonimmune handicapped is often caused long-term inapparent infection, even cause [Huang ES.The pathogenicity of Human Cytomegalovirus.Springer-Vellag.Berin, 1993, the 1-45] such as conversion, distortion or cancerations of cells infected.Therefore diagnose fast and accurately HCMV virus infection tool to be of great significance.
the cytomegalovirus infection diagnostic method has cast-off cells and tissue pathology checking at present, virus is separated, molecular hybridization test and Serological testing, but because the first three methods technical equipment requires high, the limitation that sense cycle is long, can't be widely used, and Serological testing has simple and rapid characteristic, it is used widely clinically, the HCMV proteantigen that at present most detection reagent adopts box to use is mainly to derive from viral cultures or recombinant expressed single protein fragments, perhaps reach poor specificity because proteantigen purity is low, cause false positive and reduce detection specificity, perhaps because the contained antigenic determinant of single fragment antigen albumen is less, the antibody of identifying is corresponding less, easily cause false negative and reduce detection sensitivity, although preparing detection reagent, multiple single fragment combination also can avoid the problems referred to above, but inevitable requirement is repeatedly extracted purge process, and the requirement of the purification techniques of small protein or polypeptide is higher, and working efficiency is too low.
In addition, in the world, lack at present in the situation of effectively treating virus infective medicament, become a kind of important medical intervention means by the vaccination prophylaxis of viral infections, the development of HCMV vaccine is also to carry out one of key areas of HCMV research, and an important prerequisite of vaccine research to be its proteantigen used should have high specific, strong immunogenicity and the complete antigenic determinant of trying one's best, the recombinant protein technology of therefore developing a kind of multi-antigenic determinant series connection is very necessary.
Summary of the invention
The technical problem that (one) will solve
The purpose of this invention is to provide a kind of recombinant human cytomegalovirus albumen, another object of the present invention is to provide the application of this recombinant human cytomegalovirus albumen in the preparation detection kit.
(2) technical scheme of the present invention
The invention provides a kind of recombinant DNA of encoding human cytomegalovirus albumen, its nucleotide sequence is as shown in SEQ ID NO:1.Human cytomegalic inclusion disease virus albumen by this recombinant DNA sequence coding is provided simultaneously, and its aminoacid sequence is as shown in SEQ ID NO:2.
The present invention also provides a kind of human cytomegalic inclusion disease virus protein expression carrier pET-28a-HCMV, and it is that described recombinant DNA sequence shown in SEQ ID NO:1 is inserted into the recombinant plasmid that obtains on plasmid pET-28a, and its plasmid map as shown in Figure 2.Expression vector pET-28a-HCMV is imported in intestinal bacteria, obtain expressing the engineering strain of human cytomegalic inclusion disease virus albumen.
The present invention utilizes nucleotide sequence shown in SEQ ID NO:1 to prepare human cytomegalic inclusion disease virus albumen by gene engineering method and can realize as follows:
1) obtain to have the nucleotide sequence shown in SEQ ID NO: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) cultivate described host cell under the condition that is conducive to described nucleotide sequence expression;
5) recombinant protein that recovery, purifying and renaturation are expressed.
Detection human cytomegalovirus infection's provided by the invention test kit, the antigen in its component is recombinant human herpes simplex virus albumen of the present invention.The white self-control of the marker choosing Radioactive colloidal gold, the horseradish peroxidase (HRP) that are used for mark human cytomegalic inclusion disease virus albumen.
In the test kit of employing colloid gold label antigen of the present invention, the coated concentration of anti-human IgG antibody line is 2.0mg/ml, and HCMV antigen colloidal gold mixture carries the gold pad in concentrated stoste to being coated with between diluting one times.Anti-human IgG antibody line amount is 1.0 μ l/cm, and Radioactive colloidal gold binding substances specking amount is 25.0 μ l/cm.Rabbit anti-cytomegalovirus antibody sandwich amount is 1.0 μ l/cm, and coated concentration is 5.0mg/ml.
Below with more detailed description technical scheme of the present invention:
The invention discloses a kind of nucleotide sequence as shown in SEQ ID NO:1 of encoding human cytomegalovirus albumen, utilize this nucleotide sequence to prepare the method for human cytomegalic inclusion disease virus albumen, the human cytomegalic inclusion disease virus albumen that comprises aminoacid sequence shown in SEQ ID NO:2 by the method preparation, and the composition and the test kit that comprise this albumen, they are also disclosed simultaneously in the application that detects the human cytomegalovirus infection.
Nucleotide sequence shown in SEQ ID NO:1 can be by the method preparation of this area routine, preferably according to the DNA sequence dna of purpose fragment, be the cDNA sequence of the upper purpose peptide section of HCMV pp150 and pp65 and the restriction enzyme site on plasmid, design two couples of PCR primer (P1, P2) and (P3, P4).P1 and p2 are used for the 1447th to the 2046th Nucleotide of amplification pp150, and p3 and p4 are used for the 1069th of amplification pp65 to the 1635th Nucleotide; With the restriction enzyme site of NdeI and XhoI, primer p2 and p3 order are complementary, and common corresponding to 3 ' end of the above-mentioned fragment of pp150 and 5 ' end sequence of the above-mentioned fragment of pp65 respectively for primer p1 and p4.
Primer sequence is as follows:
P1：ATCGCATATGGGTGACGGGCGGTTTGGCG
P2：GAAGAGGAAAAGCGTCGCGAGCGGCAG
P3：TCGCGACGCTTTTCCTCTTCGTCGTAGCAAACCAGCTCGT
P4：ATCGCTCGAGTTAGGGCACGTGCTGATAGCCGTGTTT
Take the human cytomegalic inclusion disease virus culture as template, with primer P1 and P2 amplification pp150 fragment, with primer P3 and P4 amplification pp65 fragment; The pp150 fragment that obtains take first round pcr amplification again and pp65 fragment add primer P1 and P4 as template, amplification pp150pp65 fragment; The goal gene recombinant fragment pp150pp65 that obtains connecting.
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 using as shown in SEQ ID NO:1 prepares human cytomegalic inclusion disease virus albumen.According to conventional methods, the nucleic acid molecule of nucleotide sequence as shown in SEQ ID NO:1 that contains encoding human cytomegalovirus albumen can be connected in an 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, the intestines such as intestinal bacteria sections bacillus.
The heterozygosis plasmid that the nucleic acid of code book invention albumen and pET-28a form has the stability of height, is conducive to protein expression of the present invention.
In one embodiment of the invention, as shown in Figure 2, preparation contains the expression construct of nucleic acid molecule and the pET-28a plasmid of nucleotide sequence shown in SEQ ID NO:1, and this construct is transformed BL21 (DE3), after the IPTG inducing culture, collect thalline.Can adopt the conventional resulting albumen of purification process purifying.
One embodiment of the invention relate to the human cytomegalic inclusion disease virus albumen with aforesaid method preparation, purifying, and this albumen has aminoacid sequence shown in SEQ ID NO:2.
One embodiment of the invention relate to composition and the test kit that comprises inventor cytomegalovirus albumen.Described composition or test kit can be prepared into and detect reagent or the kit form that the human herpes simplex vicus infects, and are used for easily and fast clinically and cytomegalovirus infection accurately.Any biological sample, as long as they contain human cytomegalic inclusion disease virus antibody, with regard to available albumen of the present invention, the composition or the test kit that comprise this albumen detect.
" test kit " described herein refers to utilize albumen of the present invention to complete that the human cytomegalovirus infection detects and reagent set that assembly is made.This test kit is used for the diagnosis human cytomegalovirus infection.Test kit of the present invention can comprise other a plurality of containers, wherein can contain respectively to detect standard substance used antibody, the enzyme of antibody or process mark, substrate or damping fluid etc.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.
At the test kit that is used for human cytomegalovirus infection's detection, human cytomegalic inclusion disease virus albumen of the present invention also can be through mark.Specifically can use the marks such as enzyme, metallo-chelate.Preferred mark enzyme for example, horseradish peroxidase, peroxidase, alkaline phosphatase etc.Preferred metallics has Radioactive colloidal gold etc.
The method of being combined with above-mentioned marker is 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
20
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.
(3) beneficial effect
Adopt the diagnostic kit of recombinant human cytomegalovirus albumen preparation provided by the invention, compare with the similar test kit on market, have the advantages such as high specificity, sensitivity height, well satisfied the needs of human cytomegalovirus infection's clinical diagnosis.HCMV proteantigen 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 HCMV vaccine development field.
Description of drawings
The gel electrophoresis figure of Fig. 1 pcr amplification product;
Fig. 2 is the structure schema of expression plasmid pET-28a-HCMV;
Fig. 3 induces rear thalline 12%SDS-PAGE electrophorogram, and wherein M represents Marker, 1 expression target protein.
Embodiment
Following examples are used for explanation the present invention, but are not used for limiting protection scope of the present invention.
The preparation of embodiment 1 cytomegalovirus albumen
1.1 the screening of cytomegalovirus Protein Epitopes and goal gene clone
Filter out the strong antigen epi-position of cytomegalovirus albumen by whole aminoacid sequences of Computer Analysis cytomegalovirus, described recombinant protein contains 189 amino acid of HCMV pp150 from 200 amino acid of the 483rd to the 682nd of N-end and pp65 from the 357th to 545 of N-ends successively from the N-end to the C-end.Wherein the DNA sequence dna of above-mentioned recombinant protein is as shown in SEQ ID No.2, and described pp150 purpose peptide segment DNA sequence is the 1447th to the 2046th Nucleotide, and pp65 purpose peptide segment DNA sequence is the 1069th to the 1635th Nucleotide.
According to the DNA sequence dna of purpose fragment, namely the cDNA sequence of the upper purpose peptide section of HCMV pp150 and pp65 and the restriction enzyme site on plasmid, design two pairs of PCR primers (P1, P2) and (P3, P4).P1 and p2 are used for the 1447th to the 2046th Nucleotide of amplification pp150, and p3 and p4 are used for the 1069th of amplification pp65 to the 1635th Nucleotide; With the restriction enzyme site of NdeI and XhoI, primer p2 and p3 order are complementary, and common corresponding to 3 ' end of the above-mentioned fragment of pp150 and 5 ' end sequence of the above-mentioned fragment of pp65 respectively for primer p1 and p4.
Primer sequence is as follows:
P1：ATCGCATATGGGTGACGGGCGGTTTGGCG
P2：GAAGAGGAAAAGCGTCGCGAGCGGCAG
P3：TCGCGACGCTTTTCCTCTTCGTCGTAGCAAACCAGCTCGT
P4：ATCGCTCGAGTTAGGGCACGTGCTGATAGCCGTGTTT
Above-mentioned primer is synthetic by (magnificent Bioisystech Co., Ltd).
Take human cytomegalic inclusion disease virus culture (Inst. of Viruses, China Preventive Medicine Science Academy gives) as template, with primer P1 and P2 amplification pp150 fragment, with primer P3 and P4 amplification pp65 fragment; The pp150 fragment that obtains take first round pcr amplification again and pp65 fragment add primer P1 and P4 as template, amplification pp150pp65 fragment; The goal gene recombinant fragment pp150pp65 that obtains connecting; Amplified production detects through agarose gel electrophoresis, and 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 Plasmid purification) reclaim target DNA, operation is undertaken by product description with DNA fast purifying test kit.
1.2 the structure of expression vector pET-28a-HCMV and evaluation
pET-28a carrier (available from magnificent Bioisystech Co., Ltd) and pcr amplification product target DNA fragment are through NdeI and XhoI double digestion, product purification (adopts TAKARA MiniBEST Plasmid purification test kit, lead to-Beijing TAKARA company available from the six directions) be connected with carrier by the T4DNA ligase enzyme, connect product and be transformed into e. coli bl21 (DE3) competence (available from magnificent Bioisystech Co., Ltd), coat dull and stereotyped upper 37 ℃ of the LB that contains kantlex and be inverted overnight incubation, select the bacterium colony of dull and stereotyped upper growth next day, alkaline lysis extracting plasmid, agarose gel electrophoresis selects suspicious recombinant plasmid to carry out pcr amplification as template, to recon plasmid that amplified production is arranged through restriction endonuclease NdeI and XhoI double digestion, identify, 3 positive recons of recon are wherein arranged.Select a positive recombinant to send the order-checking of match hundred victory companies to identify from 3 positive recombinants, result shows has inserted goal gene really on this plasmid, and direction of insertion is correct, with this recombinant plasmid called after expression plasmid pET-28a-HCMV.
1.3 express the structure of the engineering bacteria of human cytomegalic inclusion disease virus albumen
with expression plasmid pET-28a-HCMV chemical transformation ((U.S.) J. Pehanorm Brooker (JosephSambrook), (U.S.) D.W. Russell (DavidW.Russell) work, Huang Peitang etc. translate. the molecular cloning experiment guide. and Science Press, 2002.) change colon bacillus BL21 (DE3) bacterial strain (available from magnificent Bioisystech Co., Ltd) over to, coat dull and stereotyped upper 37 ℃ of the LB that contains kantlex and be inverted overnight incubation, select the bacterium colony LB culture medium culturing that contains kantlex of dull and stereotyped upper growth next day, with IPTG abduction delivering 5 hours, thalline after inducing is analyzed (the same document) with 12%SDS-PAGE, result as shown in Figure 3, determine that the bacterial strain of expressing human cytomegalic inclusion disease virus albumen is required engineering strain, preserve with Freezing Glycerine.
1.4 the preparation of recombinant human cytomegalovirus protein expression and purifying
The colon bacillus of the expression human cytomegalic inclusion disease virus albumen that inducing culture has built is used IPTG abduction delivering 5 hours, centrifugal collection thalline, add cellular lysate liquid (50mm pH 8.0Tris-Cl with 1: 10 (W/V), 50mm NaCl, 50% glycerine), add the magnetic agitation rotor to stir 30 minutes, through carrying out ultrasonic bacteria breaking (ice bath, power 200W, ultrasonic 3 seconds, interval 5 seconds, ultrasonic 80 times), (NiCl2 of 300ml 200mm crosses post to the Histidine affinity column, flow velocity 5ml/min; The 500ml level pad is washed notes, flow velocity 10ml/min; Ultrasonic precipitation after centrifugal with the dilution of 200ml level pad after loading, flow velocity 3ml/min, the 500ml level pad is washed notes, flow velocity 5ml/min; With the elution buffer wash-out that contains respectively imidazoles 20mm, 50mm, 100mm, each 100ml of 200mm, Ultraviolet Detector 280nm detects absorption value, collects elution peak; SDS-PAGE electrophoresis detection purified components) obtain the recombinant protein of purifying.
1.5 recombinant protein Western-blot checking
For verifying that restructuring HCMV type protein and anti-HCMV antiserum(antisera) are reactive and the goal gene of recombinating obtains express and have antigenicity, test with the Western-blot method.Positive serum is: 10 parts of anti-HCMV Positive Seras of rabbit (pacifying world Pharmaceutical Technology Co., Ltd available from Beijing hundred) and 30 parts of anti-HCMV Positive Seras of people (detect through the ELISA method and confirm).Negative serum is: 10 parts contrast normal rabbit serum and the 30 parts of anti-HCMV negative antibody of people serum (detect through the ELISA method and confirm).Result such as table 1.
Table 1 recombinant protein Western-blot the result
Result shows, all produce positive reaction with antigen expressed with the anti-HCMV Positive Sera of 10 portions of rabbits of HCMV viral cultures immune rabbit gained and 30 parts of anti-HCMV Positive Seras of people, 10 parts of contrast normal rabbit serums and 30 parts of people's anti-HCMV negative antibody serum and antigen expressed all produce negative reaction.Results suggest restructuring goal gene obtains to express, and restructuring HCMV protein and totivirus protein have obvious cross reactivity, and have very strong specificity, have the practical application meaning.
Preparation and the performance detecting of embodiment 2 human cytomegalic inclusion disease virus IgG antibody colloidal gold detection kit
2.1 the preparation of human cytomegalic inclusion disease virus IgG antibody colloidal gold detection kit
The above restructuring HCMV albumen that makes as the test kit labelled antigen, is measured HCMV IgG antibody with colloidal gold method.The development and application of this test kit is as follows:
(1) the present invention of test kit principle is according to immune indirect method principle, and with anti-human IgG antibody sandwich nitrocellulose filter, colloid gold label genetically engineered recombined cytomegalovirus (HCMV) antigen is tracer.Add serum to be checked during use, as containing anti-HCMV specific IgG antibodies in sample, can form mixture with the anti-human IgG antibodies on film surface, this mixture is combined with the HCMV of colloid gold label antigen and is presented the red-purple band.
(2) the test kit performance optimization (is collected many hospitals through the anti-cytomegalovirus IgG of clinical verification antibody positive with positive and negative quality control product, negative serum, recheck screening with the anti-cytomegalovirus IgG antibody ELISA test kit that Italian SORIN company provides, the anti-cytomegalovirus IgG that obtains is positive, negative serum is established as the enterprises positive and negative quality control product) be specimen, adopt the square formation titration to determine the working concentration of best anti-human IgG and HCMV antigen colloidal gold (HCMV-Ag.G) binding substances, result such as table 2, drawn by result, the coated concentration of anti-human IgG antibody line is 2.0mg/ml, the HCMV-Ag.G mixture carries the gold pad in concentrated stoste to being coated with between diluting one times.
The selection of the best anti-human IgG of table 2 and HCMV-Ag.G binding substances working concentration
-: negative reaction (without detection line); ±: probable positive (detection line mays be seen indistinctly); +: positive reaction (detection line occurring); ++: than strong positive reaction (detection line is clear); +++strong positive reaction (the detection line color is dark) (following explanation is same)
Be that 2.0mg/ml, HCMV-Ag.G mixture are concentrating stoste to coated carrying on golden basis of padding between diluting a times determining that anti-human IgG antibody line is coated with concentration, positive and negative quality control product (source and standard are the same) is as specimen take inner quality control, and the titration best anti-human IgG antibody line amount of selection (the metal spraying amount is 20 μ l/cm) is 1.0 μ l/cm.The results are shown in Table 3.
Determining of the best anti-human IgG antibody line amount of table 3
-: negative reaction; ±: probable positive; +: positive reaction; ++: than strong positive reaction; +++strong positive reaction
Determining that anti-human IgG antibody line concentration is 2.0mg/ml, the line amount is that 1.0 μ l/cm and cytomegalovirus antigen colloidal gold composite specking concentration are that concentrated stoste is to the basis between diluting a times, positive and negative quality control product (source and standard are the same) is as specimen take inner quality control, and the best Radioactive colloidal gold binding substances specking amount of employing square formation titration selection is 20.0 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; +: positive reaction; ++: than strong positive reaction; +++strong positive reaction
Select the coated concentration of nature controlling line rabbit anti-cytomegalovirus antibody on the basis of defined antigen colloidal gold composite package amount.Rabbit anti-cytomegalovirus antibody sandwich amount is 1.0 μ l/cm, and coated concentration is 3.0mg/ml.The results are shown in Table 5.
The selection of table 5 nature controlling line rabbit anti-cytomegalovirus antibody sandwich concentration
±: nature controlling line mays be seen indistinctly; +: nature controlling line appears; ++: nature controlling line is clear; +++: nature controlling line is clear thick
After anti-human IgG antibody line is coated, seal with following buffering system respectively, compare sealing effect, result shows that the rear sensitivity of closed system sealing and the specificity that contain PEG20000 all descend to some extent, other sealings and the result of not sealing 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; +: positive reaction; ++: than strong positive reaction; +++strong positive reaction
(3) preparation of test kit
1) get the HAuCl of 1.0g
4.H
2O is dissolved in the 100ml purified water, is made into 1% chlorauric acid solution; 1% chlorauric acid solution of getting 1ml enters in 100ml boiling water, adds 2ml, 1% trisodium citrate, continues to boil 30min, synthetic Radioactive colloidal gold; Get the synthetic colloidal gold solution of 6ml, carry out scanning inspection at the 400-700nm place; Get synthetic approximately 30nm Radioactive colloidal gold 406ml, use 0.1M K
2CO
3Transfer PH to 7, measure 5mg/ml 2.65ml HCMV antigen and be diluted to 40ml with the 20mM Tris-Cl of PH8.2, add while stirring colloidal gold solution, continue to stir 10min, measure 1%BSA 40ml, add while stirring aforementioned solution, continue to stir 10min, 4 ℃ of centrifugal 10min of 3000r/min, go precipitation, after the supernatant rebalancing, 4 ℃ of centrifugal 45min of 12000r/min, remove supernatant, repeat 2 times; With inner quality control serum, colloidal gold composite is tested, after the assay was approved, by aforementioned concentration specking envelope antigen colloidal gold composite, then 37 ℃, the rear cutting of the following air seasoning 16-22 of relative humidity 30% hour (spending the night).
2) precut NC film, adhesive back, the concentration of determining by the front and the coated anti-human IgG antibody of line amount and the line of rabbit anti-cytomegalovirus antibody are coated on the NC film, and drying is 1.0 hours under 37 ℃, the condition of relative humidity 30% below.
3) tear the paper film of NC film (band backboard) the detection line end of coated detection line and nature controlling line, antigen colloidal gold binding substances pad is sticked on the Quality Control line end of NC film, intersect approximately 1mm, compacting, the load sample pad that cuts out is sticked on the lower end of antigen colloidal gold binding substances pad, compacting sticks on the absorbent pad that cuts out the Quality Control line end of NC film, the check-out console two ends that post are cut remove 1cm, be cut into 4mm with slitting shear machine wide.Extract 18, detect the work in-process test card with inner quality control serum.
4) each test card is encapsulated separately, each independent packaging is 1 person-portion, and 20 person-portions are packaged into 1 box.Sampling inspection.
Each buffer formulation is as follows:
1) anti-human IgG antibody sandwich buffer formulation: 20mM Tris-Cl damping fluid (pH8.2) sees Table 7.
Table 7
2) rabbit anti-cytomegalovirus antibody sandwich buffer formulation: 20mM Tris-Cl damping fluid (pH8.2) sees Table 8.
Table 8
3) the coated buffer formulation of antigen colloidal gold mixture: 20mM TBS damping fluid (pH8.2) sees Table 9.
Table 9
(4) test kit uses working method:
1) open the packaging of aluminium foil bag of test card, take out test card.
2) test card is inclined to the application of sample nose end and is no less than 1.0cm lower than the other end.
3) get 100 μ l serum to be checked and join in the circular sample hole, room temperature (15 ℃～30 ℃) is placed and was observed and recorded result in 25 minutes.
4) assay is judged:
Positive: two red-purple lines bands appear in the interpretation window.
Negative: a red-purple lines band appears in interpretation window only nature controlling line position.
Invalid: interpretation window nature controlling line position is without red-purple lines band.
2.2 test kit Performance Detection experiment
(1) specificity (accuracy) is measured: the Radioactive colloidal gold measuring method by previous experiments is determined, detect several other serum materials, the observing response specificity.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 rubella positive serum samples, 50 parts of toxoplasma gondii 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.
(2) sensitivity determination: by the Radioactive colloidal gold measuring 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%.
(3) with the comparison test of similar products at home and abroad: comparative experiments result such as the table 10 of test kit of the present invention and 260 parts of serum samples of Italian SORIN company's test kit detection.
The compare test result of table 10 and Italian SORIN anti-cytomegalovirus IgG antibody kit
Detect total coincidence rate: (49+206)/260=98.1% shows that tentatively this test kit reaches the standard of import reagent box.
The performance detecting of embodiment 3 human cytomegalic inclusion disease virus IgG antibody assay kits (euzymelinked immunosorbent assay (ELISA))
The substrate that the restructuring HCMV albumen that embodiment 1 is made prepares as the test kit enzyme-labelled antigen is measured HCMV IgG antibody with the ELISA method.
3.1 this test kit principle and component are as follows:
1) test kit principle: the microwell plate that this strain is coated with anti-human IgG, horseradish peroxidase (HRP) marker gene engineering restructuring HCMV antigen is tracer, the TMB Color Appearance System is used the anti-cytomegalovirus II type IgG antibody in indirect method principle detection human serum or blood plasma.
2) test kit chief component composition:
Pre-coated plate, enzyme conjugates, negative control, positive control, concentrated washing lotion (20 *), substrate solution A, substrate solution B, termination liquid.
3.2 test kit performance detecting
Specificity (accuracy) is measured: press the indirect ELISA measuring method, detect several other serum materials, the observing response specificity.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 rubella positive serum samples, 50 parts of toxoplasma gondii 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 determination: press the indirect 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: press the indirect ELISA measuring method, detect the accuracy of this test kit.Use this test kit to carry out every plate diplopore to positive control, the negative control of 2 parts of anti-HCMV strong positive serum, 1 part of positive serum, 1 part of negative serum and test kit, the detection of totally 10 plates, result such as table 11, the repeatability of visible test kit is good, and the CV that different serum are detected is all lower than 15%.
The accuracy of table 11 test kit
Comparison test with similar products at home and abroad: comparative experiments result such as the table 12 of test kit of the present invention and 260 parts of serum samples of Italian SORIN company's test kit detection.
The compare test result of table 12 and Italian SORIN anti-cytomegalovirus IgG antibody kit
Detect total coincidence rate=(51+206)/260=98.8%, show that tentatively this test kit reaches the standard of import reagent box.
Claims (7)
1. encoding human cytomegalovirus protein nucleic acid is as shown in SEQ ID NO:1.
2. the human cytomegalic inclusion disease virus protein expression carrier that comprises the described nucleic acid of claim 1 is characterized in that it is that nucleic acid claimed in claim 1 is inserted into the restructuring pET-28a-HCMV plasmid that obtains on plasmid pET-28a.
3. a human cytomegalic inclusion disease virus albumen, is characterized in that it by nucleic acid encoding claimed in claim 1, and its aminoacid sequence is as shown in SEQ ID NO:2.
4. the preparation method of a recombinant human cytomegalovirus albumen, it is characterized in that application rights requires 1 recombinant DNA construction expression vector, and express the recombinant human cytomegalovirus albumen of the described recombinant DNA coding of claim 1 in the prokaryotic host cell that this carrier was fit to.
5. engineering strain of expressing human cytomegalic inclusion disease virus albumen is characterized in that it contains expression vector pET-28a-HCMV claimed in claim 3, and Host Strains is intestinal bacteria.
6. a test kit that detects the human cytomegalovirus infection, is characterized in that the antigen in its component is human cytomegalic inclusion disease virus albumen according to claim 3.
7. the application of human cytomegalic inclusion disease virus albumen according to claim 3 in the preparation detection kit.
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| 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 |
| CN103555863B (en) * | 2013-11-18 | 2015-04-01 | 瀚吉康生物科技（北京）有限公司 | Kit and method for detecting HCMV (human cytomegalovirus) |
| CN105866422B (en) * | 2015-02-09 | 2020-07-31 | 厦门大学 | Method for evaluating body's risk of developing human cytomegalovirus active infection and related kit |
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