CN102533795A - 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 fields such as genetically engineered, vaccine development and diagnostic reagent, relate to a kind of human cytomegalic inclusion disease virus albumen and application thereof particularly.
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 HCMV infects quite serious in the population of China.It is to cause that fetal in utero infects and grow damaged major reason that the women receives the HCMV primary infection at the gestation initial stage; In the fetus and newborn infant 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 also is one of organ transplantation failure and AIDS patient accompanying infection main causes of death.Non-immunodeficient person 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 the HCMV virus infection to have crucial meaning fast and accurately.
The cytomegalovirus infection diagnostic method has cast-off cells and tissue pathology checking, virus separation, molecular hybridization test and Serological testing at present; But because the first three methods technical equipment requires height; 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 detection reagent HCMV proteantigen that adopts box to use mainly is to derive from viral cultures or recombinant expressed single protein fragments mostly at present; Perhaps because proteantigen purity is low and poor specificity, causes false positive and reduce detection specificity; Perhaps because the contained antigenic determinant of single fragment antigen albumen is less; The antibody of being discerned is corresponding less; Be prone to cause false negative and reduce detection sensitivity, though multiple single fragment combination prepares detection reagent and also can avoid the problems referred to above, 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 under the situation of effectively treating virus infective medicament at present; Become a kind of important medical intervention means through the vaccination prophylaxis of viral infections; The development of HCMV vaccine also is to carry out one of key areas of HCMV research, and an important prerequisite of vaccine research to be its used proteantigen should have high specific, strong immunogenicity and the complete antigenic determinant of trying one's best, it is very necessary therefore developing the placed in-line recombinant protein technology of a kind of multi-antigenic determinant.
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 provides the application of this recombinant human cytomegalovirus albumen in the preparation detection kit.
(2) technical scheme of the present invention
The invention provides the proteic recombinant DNA of a kind of coding human cytomegalovirus, its nucleotide sequence is 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 shown in SEQ ID NO:2.
The present invention also provides a kind of human cytomegalic inclusion disease virus proteic expression vector pET-28a-HCMV, and it is that described recombinant DNA sequence shown in the SEQ ID NO:1 is inserted into the recombinant plasmid that obtains on the plasmid pET-28a, and its plasmid map is shown in accompanying drawing 2.Expression vector pET-28a-HCMV is imported in the intestinal bacteria, obtain the proteic engineering strain of expressing human cytomegalovirus.
The present invention utilizes nucleotide sequence shown in the SEQ ID NO:1 to prepare human cytomegalic inclusion disease virus albumen through gene engineering method and can realize through following steps:
1) obtains to have the nucleotide sequence shown in the 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) under the condition that helps said nucleotide sequence expression, cultivate said host cell;
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.Be used for the white self-control of the proteic affinity tag choosing of labelling human cytomegalovirus Radioactive colloidal gold, horseradish peroxidase (HRP).
In the antigenic test kit of employing colloid gold label of the present invention, it is 2.0mg/ml that anti-human IgG antibody's line encapsulates concentration, and HCMV antigen colloidal gold mixture carries the gold pad in concentrated stoste to encapsulating between diluting one times.Anti-human IgG antibody's 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 encapsulating concentration is 5.0mg/ml.
Below with more detailed description technical scheme of the present invention:
The invention discloses the proteic nucleotide sequence shown in SEQ ID NO:1 of a kind of coding human cytomegalovirus; Utilize this nucleotide sequence to prepare the proteic method of human cytomegalic inclusion disease virus; The human cytomegalic inclusion disease virus albumen that comprises aminoacid sequence shown in the SEQ ID NO:2 by this method preparation; And comprise this proteic compsn and test kit, they are also disclosed simultaneously in the application that detects the human cytomegalovirus infection.
Nucleotide sequence shown in the SEQ ID NO:1 can be through the conventional method preparation in this area; Preferably according to the segmental dna sequence dna of purpose, promptly HCMV pp150 and pp65 go up the cDNA sequence of purpose peptide section and the restriction enzyme site on the plasmid, design two couples of PCR primer (P1; P2) and (P3, P4).P1 and p2 the 1447th to the 2046th Nucleotide of pp150 that is used to increase, p3 and p4 are used to increase the 1069th of pp65 to the 1635th Nucleotide; Primer p1 and p4 have the restriction enzyme site of NdeI and XhoI respectively, and primer p2 and p3 order are complementary, and jointly corresponding to pp150 above-mentioned segmental 3 ' the terminal and above-mentioned segmental 5 ' end sequence of pp65.
Primer sequence is following:
P1：ATCGCATATGGGTGACGGGCGGTTTGGCG
P2：GAAGAGGAAAAGCGTCGCGAGCGGCAG
P3：TCGCGACGCTTTTCCTCTTCGTCGTAGCAAACCAGCTCGT
P4：ATCGCTCGAGTTAGGGCACGTGCTGATAGCCGTGTTT
With the human cytomegalic inclusion disease virus culture is template, with primer P1 and P2 amplification pp150 fragment, with primer P3 and P4 amplification pp65 fragment; The pp150 fragment and the pp65 fragment that obtain with first round pcr amplification are template again, add primer P1 and P4, amplification pp150pp65 fragment; Obtain placed in-line goal gene recombinant fragment pp150pp65.
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 nucleotide sequence of application shown in SEQ ID NO:1 and prepare the proteic method of human cytomegalic inclusion disease virus.According to conventional methods, can with contain the coding human cytomegalovirus proteic shown in SEQ ID NO:1 the nucleic acid molecule of nucleotide sequence be connected in the expression vector, then through 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 SEQ ID NO:1, and this construct is transformed BL21 (DE3), behind the IPTG inducing culture, collects 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 method for preparing, purifying, and this albumen has aminoacid sequence shown in the SEQ ID NO:2.
One embodiment of the invention relate to and comprise proteic compsn of inventor cytomegalovirus and test kit.Said compsn 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 clinically easily and fast 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, comprises this proteic compsn or test kit and detects.
" test kit " described herein is meant and utilizes albumen of the present invention to accomplish that the human cytomegalovirus infection detects and reagent set that assembly is processed.This test kit is used to diagnose the human cytomegalovirus 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, like microtiter plate etc.
Be used for the test kit that the human cytomegalovirus infection detects, human cytomegalic inclusion disease virus albumen of the present invention also can be through mark.Specifically can use marks such as enzyme, metallo-chelate.Preferred mark property enzyme for example, horseradish peroxidase, px, SEAP etc.Preferred metallics has Radioactive colloidal gold etc.
With above-mentioned affinity tag bonded method be known.When affinity tag was enzyme, its substrate and developer can be used for measuring its activity.When using horseradish peroxidase, with 3 ', 3 ', 5 ', 5 ' ,-TMB is as substrate solution, and uses the TMB Color Appearance System.When using px, with H
20
2As substrate solution, and with the amino antipyrine of O-Phenylene Diamine, 4-etc. as developer.When using SEAP, available ortho-nitrophenyl phosphoric acid, p-nitrophenyl phosphoric acid etc. are as substrate.
(3) beneficial effect
Adopt the diagnostic kit of recombinant human cytomegalovirus protein Preparation provided by the invention, compare, have advantages such as high specificity, sensitivity height, well satisfied the needs of human cytomegalovirus infection's clinical diagnosis with the similar test kit on the market.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 back thalline 12%SDS-PAGE electrophorogram, and wherein M representes Marker, 1 expression target protein.
Embodiment
Following examples are used to explain the present invention, but are not used for limiting protection scope of the present invention.
The proteic preparation of embodiment 1 cytomegalovirus
1.1 screening of cytomegalovirus proteantigen epi-position and goal gene clone
Whole aminoacid sequences through the Computer Analysis cytomegalovirus filter out the proteic strong antigen epi-position of cytomegalovirus, and described recombinant protein contains 200 amino acid and pp65 189 amino acid from N-end 357th to 545 of HCMV pp150 from terminal the 483rd to the 682nd of N-successively from the N-end to the C-end.Wherein the dna sequence dna of above-mentioned recombinant protein is 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 segmental dna sequence dna of purpose, promptly HCMV pp150 and pp65 go up the cDNA sequence and the restriction enzyme site on the plasmid of purpose peptide section, design two pairs of PCR primers (P1, P2) and (P3, P4).P1 and p2 the 1447th to the 2046th Nucleotide of pp150 that is used to increase, p3 and p4 are used to increase the 1069th of pp65 to the 1635th Nucleotide; Primer p1 and p4 have the restriction enzyme site of NdeI and XhoI respectively, and primer p2 and p3 order are complementary, and jointly corresponding to pp150 above-mentioned segmental 3 ' the terminal and above-mentioned segmental 5 ' end sequence of pp65.
Primer sequence is following:
P1：ATCGCATATGGGTGACGGGCGGTTTGGCG
P2：GAAGAGGAAAAGCGTCGCGAGCGGCAG
P3：TCGCGACGCTTTTCCTCTTCGTCGTAGCAAACCAGCTCGT
P4：ATCGCTCGAGTTAGGGCACGTGCTGATAGCCGTGTTT
Above-mentioned primer is synthetic by (magnificent Bioisystech Co., Ltd).
With human cytomegalic inclusion disease virus culture (Inst. of Viruses, China Preventive Medicine Science Academy gives) is template, with primer P1 and P2 amplification pp150 fragment, with primer P3 and P4 amplification pp65 fragment; The pp150 fragment and the pp65 fragment that obtain with first round pcr amplification are template again, add primer P1 and P4, amplification pp150pp65 fragment; Obtain placed in-line goal gene recombinant fragment pp150pp65; Amplified production detects through agarose gel electrophoresis, and the result is shown in accompanying drawing 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; Available from the six directions logical-Beijing TAKARA company) after the T4DNA ligase enzyme be 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 cultures 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, and recon plasmid that amplified production is arranged through restriction endonuclease NdeI and XhoI double digestion, evaluation, is wherein had 3 positive recons of recon.From 3 positive recombinants, select a positive recombinant to send match hundred victory companies order-checking to identify, 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-HCMV.
1.3 the structure of the proteic engineering bacteria of expressing human cytomegalovirus
With expression plasmid pET-28a-HCMV with chemical transformation ((U.S.A) J. Sa nurse Brooker (JosephSambrook); (U.S.A) D.W. Russell (DavidW.Russell) work; Huang Peitang etc. translate. the molecular cloning experiment guide. and Science Press, 2002.) change ETEC BL21 (DE3) bacterial strain (available from magnificent Bioisystech Co., Ltd) over to, coat the LB that contains kantlex and be inverted overnight cultures for dull and stereotyped last 37 ℃; Select the dull and stereotyped bacterium colony of going up growth next day with the LB culture medium culturing that contains kantlex; With IPTG abduction delivering 5 hours, the thalline after inducing was analyzed (the same document) with 12%SDS-PAGE, and the result is shown in accompanying drawing 3; Confirm that the proteic bacterial strain of expressing human cytomegalovirus is required engineering strain, with the freezing preservation of glycerine.
1.4 proteic expression preparation of recombinant human cytomegalovirus and purifying
The proteic ETEC of expressing human cytomegalovirus that inducing culture has made up with IPTG abduction delivering 5 hours, centrifugal collection thalline, adds cellular lysate liquid (50mm pH 8.0Tris-Cl with 1: 10 (W/V); 50mm NaCl, 50% glycerine), add the magnetic agitation rotor and stirred 30 minutes; Through carrying out ultrasonic bacteria breaking (ice bath, power 200W, ultrasonic 3 seconds; 5 seconds at interval, 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 deposition after centrifugal goes up appearance with 200ml level pad dilution back, flow velocity 3ml/min, and 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 purified recombinant albumen.
1.5 recombinant protein Western-blot checking
For verifying that reorganization HCMV type protein and anti-HCMV antiserum(antisera) are reactive and the goal gene of recombinating obtains expression and has antigenicity, test with the Western-blot method.Positive serum is: the 10 parts of anti-HCMV antibody positive of rabbit serum (available from Beijing hundred peace world Pharmaceutical Technology Co., Ltd) and the 30 parts of anti-HCMV antibody positive of people serum (detecting confirmation through the ELISA method).Negative serum is: 10 parts contrast normal rabbit serum and the 30 parts of anti-HCMV 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
The result shows; 10 parts of rabbit anti-HCMV antibody positive serum and the 30 parts of anti-HCMV antibody positive of people serum with HCMV viral cultures 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-HCMV negative antibody serum and antigen expressed all produce negative reaction.Results suggest reorganization goal gene obtains to express, and reorganization HCMV protein and totivirus protein have tangible cross reactivity, and have very strong specificity, have the practical application meaning.
The preparation of embodiment 2 human cytomegalic inclusion disease virus IgG antibody colloidal gold detection kit and performance calibrating
2.1 the preparation of human cytomegalic inclusion disease virus IgG antibody colloidal gold detection kit
The above reorganization HCMV albumen that makes as the test kit labelled antigen, is measured HCMV IgG antibody with colloidal gold method.The development of this test kit and using as follows:
(1) the present invention of test kit principle encapsulates nitrocellulose filter according to immune indirect method principle with anti-human IgG antibody, and 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 the sample, then can combine to form mixture with the anti-human IgG antibody on film surface, this mixture combines with the HCMV antigen of colloid gold label and presents the red-purple band.
(2) the test kit performance optimization (is collected how tame hospital through the anti-cytomegalovirus IgG of clinical verification antibody positive, negative serum with positive and negative quality control product; Anti-cytomegalovirus IgG antibody ELISA test kit with Italian SORIN company provides is rechecked screening; The anti-cytomegalovirus IgG positive, the negative serum that obtains is established as the positive and negative quality control product of enterprises) be specimen; Adopt the square formation titration to confirm 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 the result, it is that 2.0mg/ml, HCMV-Ag.G mixture carry the gold pad in concentrated stoste to encapsulating between diluting one times that anti-human IgG antibody's line encapsulates concentration.
The selection of best anti-human IgG of table 2 and HCMV-Ag.G binding substances working concentration
-: negative reaction (no 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 together)
Confirming that anti-human IgG antibody's line encapsulates concentration is that 2.0mg/ml, HCMV-Ag.G mixture are concentrating stoste to encapsulating on the basis of carrying the gold pad between diluting one times; 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-human IgG antibody's line amount (the metal spraying amount is 20 μ l/cm) is selected in titration.The result sees table 3.
Confirming of the best anti-human IgG antibody's line amount of table 3
-: negative reaction; ±: probable positive; +: positive reaction; ++: than strong positive reaction; +++strong positive reaction
Confirming that the anti-human IgG antibody concentration of ruling is 2.0mg/ml; The line amount is that 1.0 μ l/cm and cytomegalovirus antigen colloidal gold mixture 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 20.0 μ l/cm.The result sees table 4.
Confirming 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
On the basis of confirming antigen colloidal gold mixture package amount, select the concentration that encapsulates of nature controlling line rabbit anti-cytomegalovirus antibody.Rabbit anti-cytomegalovirus antibody sandwich amount is 1.0 μ l/cm, and encapsulating concentration is 3.0mg/ml.The result sees 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's line encapsulates; Seal with following buffering system respectively; Compare sealing effect; The result shows that the closed system sealing back sensitivity and the specificity that contain PEG20000 all descend to some extent, and other sealings are as broad as long with the result who does not seal, so select not seal nitrocellulose filter.The result sees 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) gets the HAuCl of 1.0g
4.H
2O is dissolved in the 100ml purified water, is made into 1% chlorauric acid solution; Get 1% chlorauric acid solution of 1ml and go in the 100ml boiling water, add 2ml, 1% trisodium citrate, continue to boil 30min, synthetic Radioactive colloidal gold; Get 6ml synthetic colloidal gold solution, carry out scanning inspection at the 400-700nm place; Get the about 30nm Radioactive colloidal gold of synthetic 406ml, use 0.1M K
2CO
3Transfer PH to 7, measure 5mg/ml 2.65ml HCMV antigen and be diluted to 40ml, add colloidal gold solution while stirring, continue stirring 10min with the 20mM Tris-Cl of PH8.2; Measure 1%BSA 40ml, add aforementioned solution while stirring, continue to stir 10min; 4 ℃ of centrifugal 10min of 3000r/min go deposition, and supernatant is again after the balance; 4 ℃ of centrifugal 45min of 12000r/min remove supernatant, repeat 2 times; With inner quality controlled serum colloidal gold composite is tested, after the assay was approved, by aforementioned concentration specking envelope antigen colloidal gold composite, then 37 ℃, relative humidity 30% following air seasoning 16-22 hour (spending the night) back cutting.
2) precut NC film, adhesive back, the concentration of confirming by the front and line amount encapsulate anti-human IgG antibody and rule with rabbit anti-cytomegalovirus antibody and encapsulate on the NC film, and drying is 1.0 hours under 37 ℃, the condition of relative humidity below 30%.
3) tear the paper film that the NC film (band backboard) that encapsulates detection line and nature controlling line detects line end, antigen colloidal gold binding substances pad is sticked on the Quality Control line end of NC film, intersect about 1mm; Compacting; With cutting out the good lower end that the appearance pad sticks on antigen colloidal gold binding substances pad of carrying, compacting is with cutting out the Quality Control line end that good absorbent pad sticks on the NC film; The check-out console two ends that post are cut remove 1cm, it is wide to be cut into 4mm with slitting shear machine.Extract 18, detect the work in-process test card with inner quality controlled 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 following:
1) anti-human IgG antibody encapsulates buffer formulation: 20mM Tris-Cl damping fluid (pH8.2) is seen table 7.
Table 7
2) rabbit anti-cytomegalovirus antibody sandwich buffer formulation: 20mM Tris-Cl damping fluid (pH8.2) is seen table 8.
Table 8
3) the antigen colloidal gold mixture encapsulates buffer formulation: 20mM TBS damping fluid (pH8.2) is seen table 9.
Table 9
(4) test kit uses working method:
1) opens the packaging of aluminium foil bag of test card, take out test card.
2) test card being inclined to the application of sample nose end is lower than the other end and is no less than 1.0cm.
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 the record 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 does not have 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 confirmed, detect several kinds of 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 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 basically, and specificity is fine.
(2) sensitivity determination:, detect the susceptibility of this test kit by the Radioactive colloidal gold measuring method that previous experiments is confirmed.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: the 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%, show that tentatively this test kit reaches the standard of import reagent box.
The performance calibrating of embodiment 3 human cytomegalic inclusion disease virus IgG antibody assay kits (ELISA)
The substrate that the reorganization 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 following:
1) test kit principle: the microwell plate that this strain encapsulates with anti-human IgG; Horseradish peroxidase (HRP) marker gene engineering reorganization HCMV antigen is tracer; The TMB Color Appearance System is used the indirect method principle and is detected the anti-cytomegalovirus II type IgG antibody in human serum or the blood plasma.
2) the main moity of test kit:
Encapsulate plate, enzyme conjugates, negative control, positive control in advance, concentrate washing lotion (20 *), substrate solution A, substrate solution B, middle solution stopping.
3.2 test kit performance calibrating
Specificity (accuracy) is measured: press the indirect ELISA measuring method, detect several kinds of 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 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 basically, 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 that 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 are carried out every plate diplopore; 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 all is lower than 15%.
The accuracy of table 11 test kit
Comparison test with similar products at home and abroad: the 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. coding human cytomegalovirus protein nucleic acid is shown in SEQ ID NO:1.
2. the proteic expression vector of human cytomegalic inclusion disease virus that comprises the said nucleic acid of claim 1 is characterized in that it is that the described nucleic acid of claim 1 is inserted into the reorganization pET-28a-HCMV plasmid that obtains on the plasmid pET-28a.
3. a human cytomegalic inclusion disease virus albumen is characterized in that it by the described nucleic acid encoding of claim 1, and its aminoacid sequence is shown in SEQ ID NO:2.
4. proteic preparation method of recombinant human cytomegalovirus; It is characterized in that application rights requires 1 recombinant DNA construction expression vector, and in the prokaryotic host cell that this carrier was fit to, express the recombinant human cytomegalovirus albumen of the said recombinant DNA coding of claim 1.
5. proteic engineering strain of expressing human cytomegalovirus is characterized in that it contains the described expression vector pET-28a-HCMV of claim 3, and the host bacterium 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 2.
7. the application of human cytomegalic inclusion disease virus albumen according to claim 2 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 |
| CN103555863A (en) * | 2013-11-18 | 2014-02-05 | 瀚吉康生物科技（北京）有限公司 | Kit and method for detecting HCMV (human cytomegalovirus) |
| 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) |
| CN105866422A (en) * | 2015-02-09 | 2016-08-17 | 厦门大学 | Method for assessing risk of body suffered from human cytomegalovirus active infection and related kit |
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| CN102533795B (en) | 2013-06-05 |
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