CN107129539B - A kind of HCV recombination fused antigens and its application - Google Patents
A kind of HCV recombination fused antigens and its application Download PDFInfo
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Abstract
The present invention relates to vitro detection diagnostic reagents, specifically disclose a kind of recombination fused antigen for being used to detect HCV, are made of Core antigens (2 102aa), flexible linker and NS5 antigens (2322 2422aa).The present invention removes C-terminal additional amino acid, selects 2 102aa antigenic domains, reduce false positive rate without damaging sensitivity by the optimization to nucleocapsid protein antigenic domains.And the 2322 2422aa amino acid regions of non-structural protein NS5 are added on the basis of nucleocapsid protein, make provided fused antigen that there is very high sensitivity, very strong specific and good thermal stability.
Description
Technical field
The present invention relates to vitro detection diagnostic reagent, specifically, being related to a kind of anti-for detecting the recombination of HCV fusion
It is former.
Background technology
Hepatitis C Virus (Hepatitis c virus, HCV) infection is great health problem, global average sense
Dye rate is about 3%.Acute infection individual more than 75% is eventually developed to chronic carrier state, eventually leads to hepatic sclerosis, liver function
It can failure and liver cancer.Hepatitis C Virus mainly by blood born, accounts for the 80-90% of post-transfusion hepatitis.HCV infection at present
Therapy is interferon and Ribavirin combination therapies, but the therapy is not all effective in all cases.
Due to effective medicine currently without hepatitis C, HCV infection, accurate judgement virus subtype and patient's infection are found as early as possible
Situation is the important means of prevention and control hepatitis C.Therefore, the good diagnostic reagent pair of high sensitivity, high specificity, stability is utilized
Blood source and blood product etc. perform stringent HCV screenings program, play an important role to effective prevention and control hepatitis C.
HCV virus belongs to the hepatitis virus category of flaviviridae, and genome is the justice of 9.6kb, single stranded RNA, encodes one
The polyprotein precursor of a 3011 amino acid.Polyprotein precursor is cut under the action of host and viral oneself protease
Multiple functional proteins, including 3 kinds of structural proteins, i.e. nucleocapsid protein Core, membrane glycoprotein E1 and envelope glycoprotein E2；And
6 kinds of non-structural proteins, i.e. NS2, NS3, NS4A, NS4B, NS5A and NS5B.
Since HCV in 1989 is found, international HCV diagnostic reagents have had evolved to the third generation.The first generation is anti-
The antigen that HCVELISA diagnostic reagents use is C100-3, and C100-3 is HCV nonstructural genes (NS3/NS4) and human superoxide
The fusion protein that mutase (SOD) gene is expressed after being fitted into, overall length are 527 amino acid, and wherein C100 accounts for 363 amino acid,
SOD accounts for 154 amino acid.After carrying out HCV screenings to blood donor using C100-3, Posttransfusion Hepatitis C incidence declines
84%.But first generation antibody has some deficiency:1st, sensitivity is low, there is false negative；2nd, specificity is low, false positive easily occurs；3rd, no
Communicable index can be used as.The blood product of only 17~25% anti-C100-3 positives can propagate HCV；4th, it should not be used in acute
The early diagnosis of hepatitis C, since later, the anti-C100-3 antibody sun of acquired hepatitis C type hepatitis patient after blood transfusion occurs in anti-C100
Property average time be 18 weeks.To make up the deficiency of first generation reagent, and second generation anti-HCV ELISA diagnostic reagents are developed.
Second generation anti-HCV ELISA diagnostic reagents include HCV nucleocapsids area recombinant protein c 22-3 and NS3 gene area recombination egg
White C33C antigens.Second generation anti-HCV ELISA diagnostic reagents, in HCV infection people at highest risk (such as Rend dialysis patient, intravenous drug
Addict and haemophiliac) in, HCV-Ab IgG recall rate is high compared with the first generation by 5~40%；In acute Posttransfusion Hepatitis C and distribute
Property hepatitis C in, HCV-Ab IgG recall rate is higher than the first generation about more than 20%, and in chronic hepatitis C, HCV-Ab IgG recall rate is compared with the
A generation increases by more than 10%.In addition, the window phase of HCV patients serums conversion is from the 18 of first generation anti-HCV ELISA diagnostic reagents
It is reduced in week 10 weeks.But there are still false positive issues and a small number of missing inspection problems for second generation anti-HCV ELISA diagnostic reagents.1996
Year, FDA had approved third generation anti-HCV ELISA reagents, compared with second generation anti-HCV ELISA reagents, increased HCV NS5
Area can detect independent NS5 antibody positives person, specificity is made to reach 99% as envelope antigen.
It is HCV3.0RIBA that generally acknowledged Hepatitis C Virus, which makes a definite diagnosis reagent, at present, which includes 2 recombinant antigens
(C33C, NS5) and 3 synthetic peptides (C100, C22,5-1-1), available for determining probable positive sample.Artificial synthetic polypeptide antigen
There is specific good, no infectivity, but there is also some shortcomings, synthesis polypeptide molecular weight is smaller, is not easy to be adsorbed to solid
Phase；Structure is shorter, low with affinity of antibody, comprising epitope it is less, missing inspection easily occurs；The cost of synthetic peptide is higher, unfavorable
In popularization etc..Other than missed detection risk, due to the main raw material(s) recombinant antigen used in Hepatitis C Virus diagnostic reagent
It is bioactive substance, activity is easily influenced by temperature and inactivates, and the thermal stability of antigen is poor, while is being prepared into HCV
It is required to after detection kit in 2-8 DEG C of preservation, it is influenced by ambient temperature also larger.In actual use, due to inspection
The preservation condition of test agent box sometimes can not be fully up to expectations, it is often desirable to which detection kit becomes by rugged environment
The stability and confidence level of height can also be kept after change.Thus, one has the HCV for reducing missed detection risk, improving heat resistance
Recombination fused antigen is the target pursued always in Hepatitis C Virus diagnosis detection.
Invention content
In order to solve the problems in the existing technology, the object of the present invention is to provide a kind of high sensitivity, high specificities
And thermal stability detects well the recombination fused antigen of HCV antibody.
In order to realize the object of the invention, technical scheme is as follows：
Present invention firstly provides a kind of HCV to recombinate fused antigen, and the fused antigen is by Core antigens (2-102aa), soft
Property connexon and NS5 antigens (2322-2422aa) form.
The fused antigen in design, first passes around the optimization to nucleocapsid protein antigenic domains, removes the extra amino of C-terminal
Acid selects 2-102aa antigenic domains, reduces false positive rate without damaging sensitivity.Then, on the basis of above-mentioned nucleocapsid protein
Add the 2322-2422aa amino acid regions of non-structural protein NS5, largely improve the fused antigen sensitivity and
Thermal stability.
Since the isoelectric point of nucleocapsid protein is very high, recombinant protein is extremely unstable under the conditions of common buffer, causes
It purifies, preserves difficulty, need to add surfactant.The addition of surfactant increases purifying cost.Meanwhile surface is lived
Property agent also has an impact HCV diagnosis systems, is easy to cause the appearance of false positive.The present invention uses HCV nucleocapsid proteins and non-knot
The method of structure albumen NS5 amalgamation and expressions, can largely improve the hydrophily and stability of recombinant antigen, and not damage recombination
The antigenicity of antigen.
Moreover, the primary structure and two of the invention according to HCV nucleocapsid proteins and non-structural protein NS5 epitopes
The characteristics of level structure selection flexible linker is attached, which also improves the hydrophily of fused antigen, and can
So that HCV nucleocapsid proteins (core antigens) and non-structural protein NS5 epitopes preferably fold to form correct space knot
Structure, significantly improves the hydrophily and stability of fused antigen, and does not damage the antigenicity of fused antigen.
More specifically, fused antigen albumen, flexible linker and SEQ ID NO.2 as shown in SEQ ID NO.1
Shown albumen is formed.
Said elements in fused antigen are not required to using the sequence normally occurred in native HCV polyprotein.Therefore, Core resists
Original can be located at N- the and/or C- ends of fusion protein.The fused antigen is followed successively by by N-terminal to C-terminal：Shown in SEQ ID NO.1
Albumen, flexible linker and albumen shown in SEQ ID NO.2；Or it is followed successively by：Albumen, flexible linker shown in SEQ ID NO.2
With albumen shown in SEQ ID NO.1.
Further, the amino acid sequence of the flexible linker is as shown in SEQ ID NO.3.
Preferably, the amino acid sequence of the fused antigen is as shown in SEQ ID NO.7.
On the basis of the above, the encoding gene of the fused antigen also belongs to protection scope of the present invention.
Further, the encoding gene of the fused antigen is by coding HCV nucleocapsid protein, flexible linker and non-structural
The nucleotide sequence composition of albumen NS5 antigens.Optionally, it is made of the nucleotide sequence shown in SEQ ID NO.4,5,6.
The nucleotide sequence of the encoding gene additionally depends in foregoing fusion albumen N-terminal to the element orders of C-terminal.
Preferably, the encoding gene is one of following (1)~(3)：
(1) nucleotide sequence shown in SEQ ID NO.8；
(2) same protein, but different from (1) the sequences due to degeneracy of genetic code are encoded with (1)；
(3) substitution, missing, the addition that one or more bases are carried out on the basis of (1) or (2) are modified, and encode
The nucleotide sequence of same protein.
It encodes obtained fusion protein and is followed successively by HCV nucleocapsid proteins, flexible linker and non-structural by N-terminal to C-terminal
Albumen NS5 antigens.
Further, recombinant vector, transgenic cell or engineering bacteria containing foregoing fusion protein coding gene also belong to
Protection scope of the present invention.
Invention further provides foregoing fusion antigen and its encoding gene answering in terms of HCV detection kits are prepared
With.
The application is converted specifically, by the encoding gene structure recombinant expression carrier of the fused antigen to engineering bacteria
Middle induced expression, obtains fused antigen.Through renaturation process after purifying, soluble fused antigen is obtained, utilizes the immune magnetic of its coating
After pearl, for detecting HCV antibody.
Empirical tests, the recombination fused antigen for preparing of the present invention with inclusion bodies despite being expressed, but very easily answer
Property, it is only necessary to conventional PBS etc. is common, and buffer can successfully obtain soluble antigen.Addition surfactant is not needed to, from
And overcome HCV nucleocapsid proteins due to must add surfactant and in the application of immune detection be limited the shortcomings that.
Therefore, reagent or kit containing fused antigen of the present invention also belong to protection scope of the present invention.
The present invention relates to raw material or reagent be ordinary commercial products, the operation being related to is unless otherwise specified
This field routine operation.
On the basis of common knowledge of the art, above-mentioned each optimum condition, can be combined with each other, obtain specific embodiment party
Formula.
The beneficial effects of the present invention are：
The present invention provides a kind of recombination fusion HCV antigens, which covers HCV nucleocapsid proteins and non-structural
The dominant antigen epitope regions of albumen NS5 have very high sensitivity, very strong specific and good thermal stability.
Meanwhile the invention discloses the HCV nucleocapsid proteins after screening and the amino acid sequence of non-structural protein NS5,
The technology path of nucleotide sequence and entire ANTIGEN DESIGNThe structure, is high-quality antibody of HCV assay kit
The exploitation of (chemoluminescence method) lays the foundation.
Description of the drawings
Fig. 1 is the digestion qualification figure of recombinant expression plasmid in the embodiment of the present invention 2；From left to right, it is followed successively by：Marker
DL10000, digestion result：pET28a-1025.
Fig. 2 is recombinant antigen induced expression qualification figure in the embodiment of the present invention 3；From left to right, it is followed successively by：Albumen
Bacterium sample after bacterium sample, induction before Marker, induction.
Fig. 3 is that recombinant antigen purifies electrophoretogram in the embodiment of the present invention 4；From left to right, it is followed successively by：Albumen Marker, bacterium
Body crushes supernatant, solubilization of inclusion bodies supernatant, penetrates sample, elution destination protein.
Fig. 4 is recombinant antigen SDS G PAGE figure in the embodiment of the present invention 4；From left to right, it is followed successively by：Albumen Marker, it answers
Protein precipitation, renaturation recombinant antigen during property.
Specific embodiment
The preferred embodiment of the present invention is described in detail below in conjunction with embodiment.It is it will be appreciated that following real
Providing merely to play the purpose of explanation for example is applied, is not used to limit the scope of the present invention.The skill of this field
Art personnel can carry out various modifications and replace to the present invention in the case of without departing substantially from spirit of the invention and spirit.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
1 HCV of embodiment recombinates the design of fused antigen
Since the antigen of viral infectious is often larger, be difficult to realize in vitro complete antigenic protein expression or
Even if realizing vivoexpression, the bigger antigen protein of molecular weight also tends to correctly to fold, and the renaturation for leading to the later stage is difficult,
Or the antigen active that renaturation obtains is very poor, constrains the exploitation of detection reagent.It therefore, will be multiple excellent using technique for gene engineering
Gesture antigen epitope genes amalgamation and expression becomes the direction of antigen research.The present invention finally determines choosing by the screening of many experiments
Two core (2-102aa), NS5 (2322-2422aa) dominant antigen epitopes are taken, pass through hydrophily after being optimized to codon
Stronger flexible connection GGGSGG is connected.The epitope of the Core of selection is 2-102aa, amino acid sequence SEQ
ID NO.1, the nucleotides sequence after corresponding optimization are classified as SEQ ID NO.4.The epitope of the NS5 of selection is 2322-
2422aa, amino acid sequence are SEQ ID NO.2, and the nucleotides sequence after corresponding optimization is classified as SEQ ID NO.5.It will be above-mentioned
Amino acid sequence after two epitopes are connected by corresponding connexon is Core+NS5 (SEQ ID NO.7), and the HCV is heavy
It is core+ns5 (SEQ ID NO.8) that group fused antigen, which corresponds to the nucleotide composition sequence after optimization,.
The expressing gene sequence of coding HCV recombination fused antigen optimized above obtains nucleosides using the method for chemical synthesis
Acid sequence core+ns5 (SEQ ID NO.8).
With core+ns5 (SEQ ID NO.8) for template, with core+ns5-F (SEQ ID NO.9), core+ns5-R
(SEQ ID NO.10) carries out PCR amplification for primer.PCR reaction systems are：
PCR reaction conditions are：94 DEG C, 5 minutes；94 DEG C, 30 seconds, 59 DEG C, 30 seconds, 72 DEG C, (amplification 30 followed within 1 minute
Ring)；72 DEG C, 5 minutes.
Amplified production is detected with 1%TAE agarose gel electrophoresis.It is recycled with a small amount of plastic recovery kits of Axygen.To obtain the final product
To target gene fragment 1025 (SEQ ID NO.11).
2 HCV of embodiment recombinates the structure of fused antigen expression vector
The target gene fragment 1025 obtained in embodiment 1 (SEQ ID NO.11) and expression vector pET28a are passed through respectively
The processing of restriction enzyme EcoRI and XhoI double digestion.
The endonuclease reaction system of target gene fragment 1025 is：
The endonuclease reaction system of expression vector pET28a is：
37 DEG C of water-bath digestions are stayed overnight.Digestion products are detected with 1%TAE agarose gel electrophoresis.It is returned with a small amount of glue of Axygen
Receive kit recycling.16 DEG C of connection 4h, and connection product is transferred to competence E.coli DH5 α cells by heat shock method, it is coated with
In containing 50 μ g/mlKan+LB agar mediums on, under the conditions of 37 DEG C, be inverted overnight incubation or culture 12~14 hours to length
Go out single bacterium colony.
It is tentatively screened using bacterium solution PCR.Specific practice is to be fallen within first with sterile toothpick picking single bacterium containing 1ml
LB(50μg/ml Kan+) 1.5ml Eppendoff pipes in, 37 DEG C of shaken cultivations take out bacterium solution after 4 hours spare.
PCR reaction systems (20 μ L) are added in following ratio：
After having added reaction system, the bacterium solutions cultivated of 1.0 μ L are drawn with small pipette tips, are mixed in mixture, after being put into PCR instrument
Start to expand.
PCR reaction conditions are：94 DEG C, 5 minutes；94 DEG C, 30 seconds, 59 DEG C, 30 seconds, 72 DEG C, (amplification 30 followed within 1 minute
Ring)；72 DEG C, 5 minutes.
Amplified production is detected with 1%TAE agarose gel electrophoresis.
It is further identified with double digestion identification.Specific practice is：It draws 10 μ L and identifies correct gram through bacterium solution PCR
Longzi bacterium solution is added to containing 5ml LB (50 μ g/ml Kan+) 15ml test tubes in, 37 DEG C of shaken cultivations are stayed overnight.Use Axygen
Mini-scale plasmid kit carries out plasmid extraction.Double digestion identification is carried out with restriction enzyme EcoRI and XhoI.
Endonuclease reaction system is：
37 DEG C of water-bath digestion 2h.Digestion products are detected with 1%TAE agarose gel electrophoresis.
To be sent to after bacterium solution PCR and plasmid enzyme restriction identify that correct bacterium solution carries out fungi preservation raw work bioengineering (on
Sea) limited company is sequenced.Sequencing result is with being expected unanimously.Base replacement, missing or addition do not occur；Double digestion
Identify the target fragment of a visible size 600bp or so, (see Fig. 1) consistent with the clip size of SEQ ID NO.8.Illustrate mesh
Gene successful clone to expression vector pET28a, be named as pET28a-1025.
3 HCV of embodiment recombinates the induced expression of fused antigen
The positive recombinant plasmid pET28a-1025 heat shock methods that correct digestion identified will be sequenced and convert Escherichia coli
E.coliBL21 (Rossetta) competent cell is coated on containing 50 μ g/ml Kan+With 25 μ g/mlChl+LB agar mediums
On, under the conditions of 37 DEG C, overnight incubation or culture 12~14 hours are inverted to growing single bacterium colony.
2 single bacterium colonies of picking are seeded to containing 50 μ g/ml Kan+With 25 μ g/mlChl+5mlLB fluid nutrient mediums in, 37
DEG C, 200r/min overnight incubations.Next day, take the switching of 200 μ l bacterium solutions in 5ml it is fresh containing 50 μ g/ml Kan+With 25 μ g/mlChl+
LB fluid nutrient mediums in, 37 DEG C, 200r/min is cultivated to OD600 ≈ 0.8.Add in IPTG to final concentration 1mM, at 37 DEG C after
Persistent oscillation culture 4 hours.SDS-PAGE electrophoresis is carried out after induction (see Fig. 2).Clone's daughter bacteria of destination protein will be induced
Liquid carries out fungi preservation, and is named as RpET28a-1025.
The clone R pET28a-1025 that selection induces destination protein are largely sent out by above-mentioned determining inductive condition
Ferment culture 2000ml.Thalline is collected, and is washed twice with the buffer solution of 20mM PBS pH7.4.RpET28a-1025 thalline freeze
In -20 DEG C.
4 HCV of embodiment recombinates the purifying of fused antigen
R pET28a-1025 fermentation thallis are resuspended with broken bacterium buffer solution (20mM PBS pH7.4,0.5M NaCl), add in
(ultrasonication condition is for ultrasonication under condition of ice bath after 1mM PMSF：Ultrasonic 5s is spaced 10s, total sonication time 15min,
It recycles 4 times altogether)；12000rpm/min after ultrasound centrifuges 15min under the conditions of 4 DEG C；Remove supernatant.With sample-loading buffer (20mM
PBS pH7.4,0.5M NaCl, 8M urea) inclusion body precipitation is resuspended, add in after 1mM PMSF ultrasonication packet under condition of ice bath
Contain body precipitation, (ultrasonication condition is：Ultrasonic 5s is spaced 10s, total sonication time 15min, recycles 2 times altogether), then 2-8
DEG C place 2-4h, inclusion body is made fully to dissolve；After solubilization of inclusion bodies, 12000rpm/min centrifuges 15min under the conditions of 4 DEG C；It takes
It is purified clearly.Sample-loading buffer is 20mMPBS pH7.4,0.5M NaCl, 8M urea；Equilibration buffer is：20mM PBS
PH7.4,0.5M NaCl, 8M urea, 30mM imidazoles；Elution buffer is：20mMPBS pH7.4,0.5M NaCl, 8M urea,
300mM imidazoles.
Broken supernatant, the sample progress SDS-PAGE electrophoresis for flowing through sample, eluting destination protein are taken respectively, and destination protein is big
Small and expected consistent (see Fig. 3), purity of protein are more than 80%, and recombinant protein is named as 1025.
5 HCV of embodiment recombinates the renaturation of fused antigen
Urea Gradient dilution refolding processing is carried out to destination protein after purification.Specially：Destination protein is packed into molecule
In the bag filter of amount 3000, the dialysis treatment in 20mM PBS pH7.4,5mM EDTA, 0.1%PC300,4M urea dialysis liquid
4h；Then destination protein is transferred in 20mM PBS pH7.4,5mM EDTA, 0.1%PC300,2M urea dialysis liquid at dialysis
Manage 4h；Then destination protein is transferred in 20mM PBS pH7.4,5mM EDTA, 0.1%PC300,1M urea dialysis liquid and is dialysed
Handle 4h；Then destination protein is transferred in 20mM PBS pH7.4,5mM EDTA, 0.1%PC300,0.5M urea dialysis liquid
Dialysis treatment 4h；Then destination protein is transferred in 20mM PBS pH7.4,5mM EDTA, 0.1%PC300 dialyzates at dialysis
4h is managed, it is primary to repeat the operation.Destination protein dialyzate is collected, 12000rpm/min centrifuges 15min under the conditions of 4 DEG C.Supernatant is
Fused antigen is recombinated for the HCV after renaturation.Sample is taken into every trade SDS-PAGE electrophoresis.Destination protein size is with expected consistent, egg
Bai Chundu is more than 80% (see Fig. 4).
6 HCV of embodiment recombinates the sensitivity technique of fused antigen
It is anti-with the HCV recombination fusions prepared by the embodiment of the present invention 5 in the case where other production technologies are completely the same
The primary antibody of HCV assay kit (chemiluminescence for Sichuan Maker Biological Science and Technology Co., Ltd.'s production
Method) in used immunomagnetic beads coating protein 1 (containing core (2-120aa), NS5 (2212-2313 aa) region it is chimeric
Antigen) it (is temporarily named as：1025 antibody of HCV assay kits (chemistry hair prepared by the HCV recombinations fused antigen
Light method)), and antibody of HCV diagnostic reagent National reference is detected, carry out the sensitivity evaluation of albumen of the present invention.
By antibody of HCV assay kit (the chemistry hair of Sichuan Maker Biological Science and Technology Co., Ltd.'s production
Light method) as contrast agents box.
The result shows that present invention recombination fused antigen detects antibody of HCV diagnostic reagent National reference
The needs of being up to state standards, meeting immune diagnostic reagent exploitation, (is shown in Table 1)
Table 1 1025 antibody of HCV assay kit (chemoluminescence method) sensitivity technique result
Note：RLU：Signal value；S/CO ＜ 1 are cloudy, and S/CO >=1 is positive.
Note：Antibody of HCV diagnostic reagent National reference, wherein N1~N30 are negative reference product；P1~P30
For positive reference product, L1~L4 is minimum detectability reference material.
Negative reference product requirement >=29/30, actually detected result are 30/30；Positive reference product requirement >=29/30, it is practical
Testing result is 29/29；Minimum detectability reference material requirement >=2/4 (L4 need to be feminine gender), actually detected result is 3/4 and L4 is
It is negative.
Contrast agents box is under 2~8 DEG C of preservation conditions, and test National reference missing inspection 4 is undesirable.
Kit containing the immunomagnetic beads for being coated with HCV recombinations fused antigen of the present invention, in 2~8 DEG C of preservation conditions
Under and after 37 DEG C accelerate heat damage 6 days, test anti-HCV National references meet the requirements.
The clinical sample detection of 7 HCV of embodiment recombination fused antigens
It is anti-with the HCV recombination fusions prepared by the embodiment of the present invention 5 in the case where other production technologies are completely the same
The primary antibody of HCV assay kit (chemiluminescence for Sichuan Maker Biological Science and Technology Co., Ltd.'s production
Method) in certain used company production immunomagnetic beads coating protein 1 (be temporarily named as：It is prepared by the HCV recombinations fused antigen
1025 antibody of HCV assay kits (chemoluminescence method)), and detect clinical random sample 1167, carry out this
The evaluation of the clinical sample coincidence rate of invention albumen.As a result, it has been found that in 1167 clinical random samples, 1025 Hepatitis C Virus
Antibody determination kit (chemoluminescence method) detects positive sample 129, Sichuan Maker Biological Science and Technology Co., Ltd.'s life
Antibody of HCV assay kit (chemoluminescence method) contrast agents of production detect positive sample 125 (tables 2).It will
Testing result inconsistent four clinical samples dj-59, dj-12, dj-64 and EPS-0358 use third party's Roche reagents respectively
It is detected, result is positive (table 3).
Table 2 1025 antibody of HCV assay kit (chemoluminescence method) clinical specificity testing result
| 1025 antibody of HCV assay kits (chemoluminescence method) | Contrast agents box | |
| Clinical sample 1167 | 129 | 125 |
The doubtful false positive serum confirmed result of 3 1025 antibody of HCV assay kit (chemoluminescence method) of table
Note：RLU：Signal value；S/CO ＜ 1 are cloudy, and S/CO >=1 is positive.
Thus illustrate, the kit containing HCV of the present invention recombination fused antigens is accurate compared to the detection of contrast agents box
True property higher.
8 HCV of embodiment recombinates the heat stability test of fused antigen
1025 immunomagnetic beads are carried out with 37 DEG C of acceleration heat damages, carries out the reagent stability evaluation of albumen of the present invention.As a result
Show that present invention recombination fused antigen thermal acceleration stability reaches requirement of the company to the product, and the immunomagnetic beads more now sold
Coating protein 1 has more preferably stability, can meet the needs of immune diagnostic reagent exploitation (table 4).
4 1025 immunomagnetic beads stability of table is verified
Note：Cal1：Negative quality-control product, Cal2：Positive quality control product, Rep1：Repeat 1, Rep2：Repeat 2, Ave：Average value
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>Sichuan mikey biology new material technology Co., Ltd
<120>A kind of HCV recombination fused antigens and its application
<130> KHP171113001.5
<160> 11
<170> PatentIn version 3.5
<210> 1
<211> 101
<212> PRT
<213> core 2-102aa
<400> 1
Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg
1 5 10 15
Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly Gly
20 25 30
Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala Thr
35 40 45
Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro Ile
50 55 60
Pro Lys Ala Arg Arg Pro Glu Gly Arg Thr Trp Ala Gln Pro Gly Tyr
65 70 75 80
Pro Trp Pro Leu Tyr Gly Asn Glu Gly Leu Gly Trp Ala Gly Trp Leu
85 90 95
Leu Ser Pro Arg Gly
100
<210> 2
<211> 101
<212> PRT
<213> NS5 2322-2422aa
<400> 2
Ala Pro Pro Ile Pro Pro Pro Arg Arg Lys Arg Thr Val Val Leu Thr
1 5 10 15
Glu Ser Thr Val Ser Ser Ala Leu Ala Glu Leu Ala Thr Lys Ala Phe
20 25 30
Gly Ser Ser Lys Ser Ser Ala Thr Asp Gly Gly Thr Ala Thr Ala Pro
35 40 45
Pro Asp His Ala Ser Asp Asp Gly Asp Lys Gly Ser Asp Val Glu Ser
50 55 60
Tyr Ser Ser Met Pro Pro Leu Glu Gly Glu Pro Gly Asp Pro Asp Leu
65 70 75 80
Ser Asp Gly Ser Trp Ser Thr Val Ser Glu Glu Ala Ser Glu Asp Val
85 90 95
Val Cys Cys Ser Met
100
<210> 3
<211> 6
<212> PRT
<213> linker
<400> 3
Gly Gly Gly Ser Gly Gly
1 5
<210> 4
<211> 303
<212> DNA
<213> core 2-102aa
<400> 4
agcacgaatc ctaaacctca aagaaaaacc aaacgtaaca ccaaccgccg cccacaggac 60
gtcaagttcc cgggcggtgg tcagatcgtt ggtggagttt acctgttgcc gcgcaggggc 120
cccaggttgg gtgtgcgcgc gactaggaag acttccgagc ggtcacaacc tcgtggaagg 180
cgacaaccta tccccaaggc tcgccgtccc gagggaagga cctgggcgca gcccgggtac 240
ccttggcccc tctatggcaa tgagggcttg gggtgggcag gatggctcct gtcaccccga 300
ggc 303
<210> 5
<211> 306
<212> DNA
<213> NS5 2322-2422aa
<400> 5
gcccccccaa taccgcctcc acggaggaag aggacggttg tcctgacaga gtccaccgtg 60
tcttctgcct tggcggagct cgctactaag gccttcggca gctccaaatc atcggccacc 120
gacggcggca cggcgaccgc ccctcctgac cacgcctccg acgacggcga caaaggatcc 180
gacgttgagt cgtactcctc catgcccccc cttgagggag agccggggga ccccgatctc 240
agcgacgggt cctggtctac cgtgagcgag gaggccagtg aggacgtcgt ctgctgctca 300
atgtaa 306
<210> 6
<211> 18
<212> DNA
<213> linker
<400> 6
ggtggtggta gtggtggt 18
<210> 7
<211> 208
<212> PRT
<213>HCV recombinates fused antigen
<400> 7
Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg
1 5 10 15
Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly Gly
20 25 30
Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala Thr
35 40 45
Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro Ile
50 55 60
Pro Lys Ala Arg Arg Pro Glu Gly Arg Thr Trp Ala Gln Pro Gly Tyr
65 70 75 80
Pro Trp Pro Leu Tyr Gly Asn Glu Gly Leu Gly Trp Ala Gly Trp Leu
85 90 95
Leu Ser Pro Arg Gly Gly Gly Gly Ser Gly Gly Ala Pro Pro Ile Pro
100 105 110
Pro Pro Arg Arg Lys Arg Thr Val Val Leu Thr Glu Ser Thr Val Ser
115 120 125
Ser Ala Leu Ala Glu Leu Ala Thr Lys Ala Phe Gly Ser Ser Lys Ser
130 135 140
Ser Ala Thr Asp Gly Gly Thr Ala Thr Ala Pro Pro Asp His Ala Ser
145 150 155 160
Asp Asp Gly Asp Lys Gly Ser Asp Val Glu Ser Tyr Ser Ser Met Pro
165 170 175
Pro Leu Glu Gly Glu Pro Gly Asp Pro Asp Leu Ser Asp Gly Ser Trp
180 185 190
Ser Thr Val Ser Glu Glu Ala Ser Glu Asp Val Val Cys Cys Ser Met
195 200 205
<210> 8
<211> 627
<212> DNA
<213>HCV recombinates fused antigen
<400> 8
agcacgaatc ctaaacctca aagaaaaacc aaacgtaaca ccaaccgccg cccacaggac 60
gtcaagttcc cgggcggtgg tcagatcgtt ggtggagttt acctgttgcc gcgcaggggc 120
cccaggttgg gtgtgcgcgc gactaggaag acttccgagc ggtcacaacc tcgtggaagg 180
cgacaaccta tccccaaggc tcgccgtccc gagggaagga cctgggcgca gcccgggtac 240
ccttggcccc tctatggcaa tgagggcttg gggtgggcag gatggctcct gtcaccccga 300
ggcggtggtg gtagtggtgg tgccccccca ataccgcctc cacggaggaa gaggacggtt 360
gtcctgacag agtccaccgt gtcttctgcc ttggcggagc tcgctactaa ggccttcggc 420
agctccaaat catcggccac cgacggcggc acggcgaccg cccctcctga ccacgcctcc 480
gacgacggcg acaaaggatc cgacgttgag tcgtactcct ccatgccccc ccttgaggga 540
gagccggggg accccgatct cagcgacggg tcctggtcta ccgtgagcga ggaggccagt 600
gaggacgtcg tctgctgctc aatgtaa 627
<210> 9
<211> 27
<212> DNA
<213>Artificial sequence
<400> 9
gaattcagca cgaatcctaa acctcaa 27
<210> 10
<211> 32
<212> DNA
<213>Artificial sequence
<400> 10
ctcgagttac attgagcagc agacgacgtc ct 32
<210> 11
<211> 639
<212> DNA
<213>Target gene fragment 1025
<400> 11
gaattcagca cgaatcctaa acctcaaaga aaaaccaaac gtaacaccaa ccgccgccca 60
caggacgtca agttcccggg cggtggtcag atcgttggtg gagtttacct gttgccgcgc 120
aggggcccca ggttgggtgt gcgcgcgact aggaagactt ccgagcggtc acaacctcgt 180
ggaaggcgac aacctatccc caaggctcgc cgtcccgagg gaaggacctg ggcgcagccc 240
gggtaccctt ggcccctcta tggcaatgag ggcttggggt gggcaggatg gctcctgtca 300
ccccgaggcg gtggtggtag tggtggtgcc cccccaatac cgcctccacg gaggaagagg 360
acggttgtcc tgacagagtc caccgtgtct tctgccttgg cggagctcgc tactaaggcc 420
ttcggcagct ccaaatcatc ggccaccgac ggcggcacgg cgaccgcccc tcctgaccac 480
gcctccgacg acggcgacaa aggatccgac gttgagtcgt actcctccat gccccccctt 540
gagggagagc cgggggaccc cgatctcagc gacgggtcct ggtctaccgt gagcgaggag 600
gccagtgagg acgtcgtctg ctgctcaatg taactcgag 639
Claims (10)
1. a kind of HCV recombinates fused antigen, which is characterized in that the fused antigen albumen, flexibility as shown in SEQ ID NO.1 connect
Albumen shown in son and SEQ ID NO.2 is connect to form.
2. fused antigen according to claim 1, which is characterized in that the fused antigen is followed successively by by N-terminal to C-terminal：SEQ
Albumen shown in ID NO.1, flexible linker and albumen shown in SEQ ID NO.2；Or it is followed successively by：Albumen shown in SEQ ID NO.2,
Albumen shown in flexible linker and SEQ ID NO.1.
3. fused antigen according to claim 2, which is characterized in that the amino acid sequence of the flexible linker such as SEQ
Shown in ID NO.3.
4. fused antigen according to claim 3, which is characterized in that the amino acid sequence of the fused antigen such as SEQ ID
Shown in NO.7.
5. the encoding gene of any one of Claims 1 to 4 fused antigen.
6. encoding gene according to claim 5, which is characterized in that it is as the nucleotide shown in SEQ ID NO.4,5,6
Sequence forms.
7. encoding gene according to claim 6, which is characterized in that it is one of following (1)~(2)：
(1) nucleotide sequence shown in SEQ ID NO.8；
(2) same protein, but different from (1) the sequences due to degeneracy of genetic code are encoded with (1).
8. recombinant vector or engineering bacteria containing any one of claim 5~7 encoding gene.
9. reagent or kit containing Claims 1 to 4 any one of them fused antigen.
10. prepared by any one of Claims 1 to 4 any one of them fused antigen or claim 5~7 encoding gene
Application in terms of HCV detection reagents or kit.
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| CN108196069B (en) * | 2018-02-01 | 2020-06-09 | 深圳德睿生物科技有限公司 | Hepatitis C virus antibody detection reagent containing recombinant fusion antigens A and B, application of hepatitis C virus antibody detection reagent and recombinant fusion antigens A and B |
| BR102018071672A2 (en) * | 2018-10-22 | 2021-11-16 | Fundação Oswaldo Cruz | POLYPEPTIDE, EXPRESSION CASSETTE, EXPRESSION VECTOR, HOST CELL, KIT FOR IMMUNOLOGICAL SCREENING OF HCV AND/OR DIAGNOSIS OF HEPATITIS C, COMPOSITION, USE OF AT LEAST ONE POLYPEPTIDE, AND, METHODS FOR PRODUCING A HCV, PARAPEPTIDE FOR THE DIAGNOSIS OF HEPATITIS C |
| CN111704657B (en) * | 2020-06-16 | 2021-11-09 | 四川安可瑞新材料技术有限公司 | HIV-2 recombinant antigen and preparation method and application thereof |
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| CN101748151A (en) * | 2008-12-19 | 2010-06-23 | 深圳市源兴生物医药科技有限公司 | Recombinant human hepatitis C virus antigen adenoviral vector and applications thereof |
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| CN101748151A (en) * | 2008-12-19 | 2010-06-23 | 深圳市源兴生物医药科技有限公司 | Recombinant human hepatitis C virus antigen adenoviral vector and applications thereof |
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