CN107098980A - A kind of fusion protein for detecting Detecting Rubella Virus Antibodies In Human Sera and preparation method thereof - Google Patents
A kind of fusion protein for detecting Detecting Rubella Virus Antibodies In Human Sera and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to Biological Detection technical field, a kind of fusion protein for detecting Detecting Rubella Virus Antibodies In Human Sera is specifically provided, fusion protein is the fusion protein comprising rubella virus E 1 protein fragment I, rubella virus E 1 protein fragment II and rubella virus C protein fragment.Present invention also offers the preparation method of fusion protein.Fusion protein of the present invention has hypersensitivity and specificity, and it is used for detect that Detecting Rubella Virus Antibodies In Human Sera is greatly improved verification and measurement ratio.
Description
Technical field
The present invention relates to technique for gene engineering and diagnostic reagent field, and in particular to one kind is used to detect Detecting Rubella Virus Antibodies In Human Sera
Fusion protein and preparation method thereof.
Background technology
Rubella virus (rubella virus, RV) is the unique member of Togaviridae rubella virus genus, unique natural
Host is people, but laboratory can and experimental animal wild with infected monkey, mouse etc..Rubella is a kind of disease as mild as a dove, generally
Only cause fash, submandibular lymph nodes enlargement and other slight signs.But to children and pregnant woman, the consequence of rubella is very serious.It is pregnant
Infection in 12 weeks can cause the congenital rubella-infection of fetus and 80-90% patient pregnant woman to miscarry before phase.The congenital infection meeting of fetus
Cause congenital rubella syndrome (CRS), involve multiple organ multisystem, and there is long-term active infection.
Rubella virus genome group be single-stranded positive RNA, structural proteins during rubella virus has 3, be respectively after birth glycoprotein E 1,
E2 and nucleocapsid protein C.C protein Pro-rich and arginine, nucleocapsid is bonded with viral RNA, in virus from a place
Chief cell shields during infecting another host cell to viral genome.E1 and E2 are present in peplos table
Face, the epitope of corresponding immune response is produced containing energy stimulation of host.E1 is the important antigen of rubella virus, although whole ammonia
There is some difference between different strains for base acid sequence, but is conservative consistent in antigenic determinant concentrated area.E2
So far not yet clearly, in E1-E2 compounds, E2 epitopes may be covered the biology of albumen by E1, so in rubella virus
The immune response of induced low levels in infection.
Rubella virus infection clinical symptoms and other viral infection symptoms have certain similitude, so clinical diagnosis is accurate
Property adds some difficulty.Rubella virus infection diagnosis is main based on serodiagnosis, and the purifying of cell culture antigen is difficult to
Cell protein composition is excluded, antigen purity is not high to cause diagnostic sensitivity and specificity poor, so screening hypersensitivity and spy
The recombinant antigen of the opposite sex is the key for developing rubella virus diagnosis.
The content of the invention
It is an object of the invention to be：In view of the deficienciess of the prior art, provide a kind of hypersensitivity, high specific,
The fusion protein of the detection Detecting Rubella Virus Antibodies In Human Sera of verification and measurement ratio can be improved.
To achieve these goals, the technical scheme is that：
It is a kind of detect Detecting Rubella Virus Antibodies In Human Sera fusion protein, the fusion protein be comprising rubella virus E 1 protein fragment I,
Rubella virus E 1 protein fragment II and rubella virus C protein fragment fusion protein, the E1 protein fragments I are distributed in fusion egg
White N-terminal, the E1 protein fragments II is distributed in centre, and the C protein fragment is distributed in the C-terminal of fusion protein, and the E1
Connected between protein fragments I, E1 protein fragments II and C protein fragment by Gly-Gly-serine.
As an improvement technical scheme, the E1 protein fragments I be in E1 protein fragments the 9th amino acid to the
116 amino acid, the E1 protein fragments II is the 208th amino acid in E1 protein fragments to the 293rd amino acid, the C
Protein fragments are the 212nd amino acid in C protein fragment to the 243rd amino acid.
As an improvement technical scheme, the E1 protein fragments I has SEQ ID NO:Amino acid sequence shown in 1
Row, the E1 protein fragments II has SEQ ID NO:Amino acid sequence shown in 2, the C protein fragment has SEQ ID
NO:Amino acid sequence shown in 3.
As an improvement technical scheme, the fusion protein has SEQ ID NO:Amino acid sequence shown in 4.
As an improvement technical scheme, the gene order such as SEQ ID NO of the fusion protein:Shown in 5, the base
Because there are two kinds of restriction enzymes of NCOI and NdeI sequence rear and front end respectively.
It is another object of the present invention to be：In view of the deficienciess of the prior art, providing a kind of detection rubella virus
The preparation method of the fusion protein of antibody.
To achieve these goals, the technical scheme is that：
A kind of preparation method for the fusion protein for detecting Detecting Rubella Virus Antibodies In Human Sera, it is characterised in that under the preparation method includes
Row step：
(1) screening of epitope and the chemical synthesis of genetic fragment
Using on-line analysis tool analysis, the 9th amino acid of rubella virus E 1 protein is filtered out to the 116th amino
Acid, the 208th amino acid of E1 albumen are to 293 amino acid and the 212nd amino acid of rubella virus C protein to the 243rd
Amino acid；Chemical synthesis rubella virus E 1 protein fragment I, E1 protein fragments II and the gene order of C protein fragment series connection；
(2) structure of expression E1 protein fragments I, E1 protein fragments II and the fusion protein recombinant plasmid of C protein fragment
PGEM-5zf is extracted, with Ncol and Ndel double digestions, the plasmid fragments of glue reclaim double digestion after electrophoresis；With Ncol and
The rubella virus fusion protein gene fraction of Ndel double digestion chemical syntheses, electrophoresis reclaims the genetic fragment of double digestion, and -20 DEG C standby
With；The genetic fragment of the plasmid fragments of double digestion and double digestion is pressed 1:3-10 ratios, with T4 ligases, 16 DEG C connect overnight, even
Recombinant plasmid after connecing is PGEM-5zf-E1+EII+C；
(3) screening and identification of recombinant plasmid
Recombinant plasmid transformed e. coli bl21 (DE3) is coated with the LB flat boards containing ampicillin, 37 DEG C of constant temperature trainings
Foster case is stayed overnight；Next day random picking conversion bacterium colony and control bacterium colony, extract plasmid respectively, respectively with Ndel and Ncol double digestions,
Electrophoresis is run after digestion, corresponding purpose fragment and carrier, as construction of expression vector success, positive expression bacterium can be seen；
As an improvement technical scheme, the preparation method also comprises the following steps：
Recombinant protein engineering bacteria high efficient expression：
Positive expression bacterium is seeded in the LB culture mediums containing ampicillin, 37 DEG C of constant-temperature tables vibrate 4h, plus dense eventually
Spend for 0.5-1.5mmol/lIPTG, continue to induce 3-4h under the conditions of 37 DEG C, precipitation thalline is collected by centrifugation, precipitation thalline is carried out
It is broken, take supernatant precipitation to carry out the destination protein expressed in SDS-PAGE detections, supernatant respectively；
The purifying of expressing protein：
Recombinant protein engineering bacteria is centrifuged into 8-12min under the conditions of 4 DEG C with 8000-12000rpm rotating speed, by precipitation
Thalline is resuspended in 1/10 lysate of former centrifugation volume, ice-bath ultrasonic thalline, with 10000-13000rpm's under the conditions of 4 DEG C
Rotating speed centrifuges 25-35min, collects bacterium solution supernatant；Rinsed with equilibrium liquid after balance nickel ion affinity chromatograph post, ultrasound is collected
Pillar is washed with equilibrium liquid after the direct upper prop of bacterium solution supernatant, end of the sample, elution albumen is used successively, each eluting peak is collected
Albumen, each peak albumen of SDS-PAGE electrophoresis detections.
As an improvement technical scheme, lysate in the purification step of expressing protein by 50mMTris-Hcl,
10mM EDTA, 15mM Nacl, 10mM DTT compositions.
As an improvement technical scheme, the miaow that the eluent in the purification step of expressing protein is concentration 20-500mM
Azoles eluent.
The present invention uses above technical scheme, compared with prior art, with advantages below：
(1) analysis E1 albumen and C protein amino acid sequence, select the stronger partial segments of antigenicity, substantially increase
The yield and specificity of expression, substantially increase clinical loss；
(2) expression E1 protein fragments I, E1 protein fragments II and the fusion protein of C protein fragment are used as detection antigen, effectively
The detection antigen for recombinating E1 Partial Proteins and C protein respectively in the prior art is solved, several tables are prepared during production simultaneously
The problem of up to the consuming time present in albumen and cost；
(3) expression rubella virus E 1 protein fragment I, E1 protein fragments II and the fusion protein of C protein fragment are with solvable shape
Formula is present, it is easy to purify.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, the present invention is carried out further detailed
Explanation.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not intended to limit the present invention.
Embodiment 1
A kind of fusion protein for detecting Detecting Rubella Virus Antibodies In Human Sera, fusion protein is to include rubella virus E 1 protein fragment I, rubella
Viral E1 protein fragments II and rubella virus C protein fragment fusion protein.Wherein E1 protein fragments I is distributed in fusion protein
Aminoterminal, E1 protein fragments II is distributed in centre, and C protein fragment is distributed in the C-terminal of fusion protein, and E1 protein fragments I, E1 eggs
Connected between white tiles section II and C protein fragment by Gly-Gly-serine.Wherein E1 protein fragments I is E1 albumen flakes
9 amino acid of Duan Zhong are to the 116th amino acid, and E1 protein fragments II is the 208th amino acid in E1 protein fragments to the 293rd
Individual amino acid, C protein fragment is the 212nd amino acid in C protein fragment to the 243rd amino acid.
Embodiment 2
A kind of preparation method for the fusion protein for detecting Detecting Rubella Virus Antibodies In Human Sera, comprises the following steps：
(1) screening of epitope and the chemical synthesis of genetic fragment
Using the on-line analysis tool analysis such as ExPasy, TMHMM, SignalIP4.1, filter out rubella virus E 1 protein and
C protein amino acid sequence total length, filters out rubella virus E 1 protein fragment I and (contains in E1 albumen compared with the 9th of strong antigen determinant
Individual amino acid is to the 116th amino acid), E1 protein fragments II (contain the 208th amino compared with strong antigen determinant in E1 albumen
Acid is to 293 amino acid) and rubella virus C protein fragment (contain the 212nd amino acid compared with strong antigen determinant in C protein
To the 243rd amino acid)；Chemical synthesis rubella virus E 1 protein fragment I, E1 protein fragments II and the base of C protein fragment series connection
Because of sequence；
(2) structure of expression E1 protein fragments I, E1 protein fragments II and the fusion protein recombinant plasmid of C protein fragment
PGEM-5zf is extracted, with Ncol and Ndel double digestions, the plasmid fragments of glue reclaim double digestion after electrophoresis；With Ncol and
The rubella virus fusion protein gene fraction of Ndel double digestion chemical syntheses, electrophoresis reclaims the genetic fragment of double digestion, and -20 DEG C standby
With；The genetic fragment of the plasmid fragments of double digestion and double digestion is pressed 1:3-10 ratios, with T4 ligases, 16 DEG C connect overnight, even
Recombinant plasmid after connecing is PGEM-5zf-E1+EII+C；
(3) screening and identification of recombinant plasmid
By recombinant plasmid transformed e. coli bl21 (DE3), coating contains on the LB flat boards of ampicillin (60ug/ml),
37 DEG C of constant incubators are stayed overnight；Next day random picking conversion bacterium colony and control bacterium colony (plasmid PGEM-5zf transformed bacterias), are carried respectively
Plasmid is taken, respectively with Ndel and Ncol double digestions, electrophoresis is run after digestion, corresponding purpose fragment and carrier can be seen, built
Expression vector success, as positive expression bacterium；
(4) recombinant protein engineering bacteria high efficient expression
In the test tube that positive expression bacterium is seeded to 2mlLB culture mediums (60ug/ml ampicillins), 37 DEG C of constant-temperature tables
4h, plus final concentration of 0.5mmol/l IPTG are vibrated, 37 DEG C are continued to induce 6h, and precipitation thalline is collected by centrifugation, and precipitation thalline is entered
Row is broken, takes supernatant precipitation to carry out the destination protein expressed in SDS-PAGE detections, supernatant respectively；
(5) purifying of expressing protein
The recombinant protein engineering bacteria single bacterium colony of high efficient expression is selected, the triangular flask of the fluid nutrient medium containing 100mlLB is inoculated into
In, plus ampicillin is put in 37 DEG C of shaking tables overnight to final concentration 60ug/ml.Next day presses bacterium solution and LB culture mediums 1:10 inoculations
Into the triangular flask of 1000mlLB (ampicillin 60ug/ml) nutrient solution, 4h, plus IPTG (final concentration of 0.5mmol/ are cultivated
L), continue under the conditions of 37 DEG C to induce 6h, the 1000ml engineering bacterias of induced expression fusion protein are centrifuged, (10000rpm) is low at a high speed
(4 DEG C) centrifugation 10min of temperature, by the thalline of precipitation be resuspended in former centrifugation volume 1/10 lysate (50mMTris-Hcl,
10mMEDTA, 15mMNaCL, 10mMDTT) in, ice-bath ultrasonic thalline, (4 DEG C) centrifugation 30min of (12000rpm) low temperature, are received at a high speed
Collect bacterium solution supernatant.
Rinsed with equilibrium liquid (20mM PB PH7.4) after balance nickel ion affinity chromatograph post, in the bacterium solution that ultrasound is collected
Wash pillar with equilibrium liquid after clear directly upper prop, loading flow velocity 1.5ml/min, end of the sample, successively with containing 20,50,100,
200th, the elution albumen of 500mM concentration imidazoles, collects each eluting peak albumen, each peak albumen of SDS-PAGE electrophoresis detections.
Embodiment 3
Application of the recombinant protein of purifying in detection Detecting Rubella Virus Antibodies In Human Sera
By the recombinant protein of purifying, 100ul/ holes after being diluted with carbonate buffer solution (50mM, PH9.6), 100ng/ holes
Protein content is coated with elisa plate, and 4 DEG C overnight.Add the PBS37 DEG C of water-bath closing 2h of the calf serum of 200ul/ holes 10%, washing after washing
Afterwards 4 DEG C it is standby.
Dot-ELISA detects rubella virus positive serum and normal human serum, as a result shows that fusion protein can be with
Rubella virus positive serum reacts, and is not reacted with normal human serum, illustrates that the fusion protein has preferably antigenicity and special
Property.
Embodiment 4
The preparation of the polyvalent antibody of rubella virus fusion protein
The recombinant rubella virus fusion protein of purifying is prepared into polyvalent antibody as antigen, it is new with antigen and adjuvant combined immunization
The mode of western orchid large ear rabbit, prepares and purifies the rabbit anti-serum of acquisition, indirect elisa method detection rubella virus antibody drop
Degree.
(1) main agents：Complete Freund's adjuvant and incomplete Freund's adjuvant are SIGMA Products.Other reagents are state
Production or Import Analysis pure reagent.
(2) immune programme for children
5 new zealand rabbits, every immune 200ug antigens are immunized as immunogene in fusion protein, and immunization wayses are the back of the body
Portion's multiple spot is immunized.It is immune for the first time to use complete Freund's adjuvant and fusion protein mixing and emulsifying；It is immune for the second time after two weeks, with not
Complete Freund's adjuvant and fusion protein mixing and emulsifying, immunization wayses are immune for back multiple spot；It is immunized, uses for the third time after three days
100ug fusion protein and 0.01MPBS (PH7.4) mixing of same volume, immunization wayses peritoneal immunity；Equally exempt from after three days
Epidemic disease program immunity is once.
(3) indirect ELISA surveys rabbit anti-serum potency
Three days after last time is immune, rabbit auricular vein takes blood, and 4000rpm centrifugations 10min obtains rabbit anti-serum.Take fusion
100ul/ holes, the protein content in 100ng/ holes are coated with elisa plate, 4 DEG C of mistakes after albumen is diluted with carbonate buffer solution (50mM, PH9.6)
Night.The calf serum of next day 20% is closed, and 2h is closed in 37 DEG C of water-baths, and using rabbit anti-serum gradient dilution as primary antibody, 37 DEG C of water-baths are incubated
Educate after 1h, plus 1:5000 secondary antibody goat-anti rabbits, plus nitrite ion colour developing.Potency up to 1:More than 10000, illustrate that fusion protein has preferably
Antigenic and specificity.
This patent is not limited to above-mentioned specific embodiment, one of ordinary skill in the art from above-mentioned design,
Without performing creative labour, made a variety of conversion are all fallen within the protection domain of this patent.
SEQUENCE LISTING
<110>Weifang Hightop Biotech Co., Ltd.
<120>A kind of fusion protein for detecting Detecting Rubella Virus Antibodies In Human Sera and preparation method thereof
<130> 1
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 107
<212> PRT
<213>E1 protein fragments 1
<400> 1
Thr Ala Pro Gly Cys Ala Thr Gln Thr Pro Val Pro Val Arg Leu Ala
1 5 10 15
Gly Val Arg Phe Glu Ser Lys Ile Val Asp Gly Gly Cys Phe Ala Pro
20 25 30
Trp Asp Leu Glu Ala Thr Gly Ala Cys Ile Cys Glu Ile Pro Thr Asp
35 40 45
Val Ser Cys Glu Gly Leu Gly Ala Trp Val Pro Thr Ala Pro Cys Ala
50 55 60
Arg Ile Trp Asn Gly Thr Gln Arg Ala Cys Thr Phe Trp Ala Val Asn
65 70 75 80
Ala Tyr Ser Ser Gly Gly Tyr Ala Gln Leu Ala Ser Tyr Phe Asn Pro
85 90 95
Gly Gly Ser Tyr Tyr Lys Gln Tyr His Pro Thr
100 105
<210> 2
<211> 85
<212> PRT
<213>E1 protein fragments II
<400> 2
Ala Gly Gly Ser Met Asn Tyr Thr Gly Asn Gln Gln Ser Arg Trp Gly
1 5 10 15
Leu Gly Ser Pro Asn Cys His Gly Pro Asp Trp Ala Ser Pro Val Cys
20 25 30
Gln Arg His Ser Pro Asp Cys Ser Arg Leu Val Gly Ala Thr Pro Glu
35 40 45
Arg Pro Arg Leu Arg Leu Val Asp Ala Asp Asp Pro Leu Leu Arg Thr
50 55 60
Ala Pro Gly Pro Gly Glu Val Trp Val Thr Pro Val Ile Gly Ser Gln
65 70 75 80
Ala Arg Lys Cys Gly
85
<210> 3
<211> 40
<212> PRT
<213>C protein fragment
<400> 3
Leu His Ile Arg Ala Gly Gly Ser Pro Ala Gly Asp Val Arg Gly Val
1 5 10 15
Trp Gly Lys Gly Glu Arg Thr Tyr Thr Glu Gln Asp Phe Arg Val Gly
20 25 30
Gly Thr Arg Trp His Arg Leu Leu
35 40
<210> 4
<211> 232
<212> PRT
<213>Fusion protein
<400> 4
Thr Ala Pro Gly Cys Ala Thr Gln Thr Pro Val Pro Val Arg Leu Ala
1 5 10 15
Gly Val Arg Phe Glu Ser Lys Ile Val Asp Gly Gly Cys Phe Ala Pro
20 25 30
Trp Asp Leu Glu Ala Thr Gly Ala Cys Ile Cys Glu Ile Pro Thr Asp
35 40 45
Val Ser Cys Glu Gly Leu Gly Ala Trp Val Pro Thr Ala Pro Cys Ala
50 55 60
Arg Ile Trp Asn Gly Thr Gln Arg Ala Cys Thr Phe Trp Ala Val Asn
65 70 75 80
Ala Tyr Ser Ser Gly Gly Tyr Ala Gln Leu Ala Ser Tyr Phe Asn Pro
85 90 95
Gly Gly Ser Tyr Tyr Lys Gln Tyr His Pro Thr Ala Gly Gly Ser Met
100 105 110
Asn Tyr Thr Gly Asn Gln Gln Ser Arg Trp Gly Leu Gly Ser Pro Asn
115 120 125
Cys His Gly Pro Asp Trp Ala Ser Pro Val Cys Gln Arg His Ser Pro
130 135 140
Asp Cys Ser Arg Leu Val Gly Ala Thr Pro Glu Arg Pro Arg Leu Arg
145 150 155 160
Leu Val Asp Ala Asp Asp Pro Leu Leu Arg Thr Ala Pro Gly Pro Gly
165 170 175
Glu Val Trp Val Thr Pro Val Ile Gly Ser Gln Ala Arg Lys Cys Gly
180 185 190
Leu His Ile Arg Ala Gly Gly Ser Pro Ala Gly Asp Val Arg Gly Val
195 200 205
Trp Gly Lys Gly Glu Arg Thr Tyr Thr Glu Gln Asp Phe Arg Val Gly
210 215 220
Gly Thr Arg Trp His Arg Leu Leu
225 230
<210> 5
<211> 708
<212> DNA
<213>Fusion protein
<400> 5
ccatggactg caccggggtg cgccacccaa acacctgtcc ccgtgcgcct cgctggcgtc 60
cgctttgagt ccaagattgt ggacggcggc tgctttgccc catgggacct cgaggccact 120
ggagcctgca tttgcgagat ccccactgac gtctcgtgcg agggcctggg ggcctgggta 180
cccacagccc cttgcgcgcg catctggaac ggcacacagc gcgcgtgcac cttctgggct 240
gtcaacgcct actcctctgg tgggtacgcg cagctggcct cctacttcaa ccctggcggc 300
agctactaca agcagtacca ccctaccgcg ggaggttcga tgaattacac tggcaaccag 360
cagtcccggt ggggcctcgg gagcccgaac tgccacggcc ccgattgggc ctccccggtt 420
tgtcaacgcc actcccctga ctgctcgcgg cttgtggggg ccacgccaga gcgtccccgg 480
ctgcgcctgg tcgacgccga cgaccccctg ctgcgcactg cccctgggcc cggcgaggtg 540
tgggtcacgc ctgtcatagg ctctcaggcg cgcaagtgcg gactccacat acgcgccgga 600
ggttcgcctg ccggcgacgt caggggcgtt tggggtaaag gcgagcgcac ctacgccgag 660
caggacttcc gcgtcggcgg cacgcgctgg caccgactgc tgcatatg 708
Claims (9)
1. a kind of fusion protein for detecting Detecting Rubella Virus Antibodies In Human Sera, it is characterised in that：The fusion protein is to include rubella virus E 1
Protein fragments I, rubella virus E 1 protein fragment II and rubella virus C protein fragment fusion protein, wherein the E1 albumen flakes
Section I is distributed in the N-terminal of fusion protein, and the E1 protein fragments II is distributed in the centre of fusion protein, the C protein fragment distribution
Pass through glycine-sweet ammonia between the C-terminal of fusion protein, and E1 protein fragments I, E1 protein fragments II and C protein fragment
Acid-serine connection.
2. a kind of fusion protein for detecting Detecting Rubella Virus Antibodies In Human Sera according to claim 1, it is characterised in that：The E1 albumen
Fragment I is the 9th amino acid in E1 protein fragments to the 116th amino acid, and the E1 protein fragments II is in E1 protein fragments
208th amino acid to the 293rd amino acid, the C protein fragment is the 212nd amino acid in C protein fragment to the 243rd
Amino acid.
3. a kind of fusion protein for detecting Detecting Rubella Virus Antibodies In Human Sera according to claim 1, it is characterised in that：The E1 albumen
Fragment I has SEQ ID NO:Amino acid sequence shown in 1, the E1 protein fragments II has SEQ ID NO:Ammonia shown in 2
Base acid sequence, the C protein fragment has SEQ ID NO:Amino acid sequence shown in 3.
4. a kind of fusion protein for detecting Detecting Rubella Virus Antibodies In Human Sera according to claim 1, it is characterised in that：The fusion egg
There is SEQ ID NO in vain:Amino acid sequence shown in 4.
5. a kind of fusion protein for detecting Detecting Rubella Virus Antibodies In Human Sera according to claim 1, it is characterised in that：The fusion egg
White gene order such as SEQ ID NO:Shown in 5, there are two kinds of limitations of NCO I and Nde I the gene order rear and front end respectively
Property restriction endonuclease.
6. a kind of preparation method for the fusion protein for detecting Detecting Rubella Virus Antibodies In Human Sera, it is characterised in that the preparation method includes following
Step：
(1) screening of epitope and the chemical synthesis of genetic fragment
Using on-line analysis tool analysis, the 9th amino acid of rubella virus E 1 protein is filtered out to the 116th amino acid, E1
208th amino acid of albumen is to 293 amino acid and the 212nd amino acid of rubella virus C protein to the 243rd amino
Acid；Chemical synthesis rubella virus E 1 protein fragment I, E1 protein fragments II and the gene order of C protein fragment series connection；
(2) structure of expression E1 protein fragments I, E1 protein fragments II and the fusion protein recombinant plasmid of C protein fragment
PGEM-5zf is extracted, with Ncol and Ndel double digestions, the plasmid fragments of glue reclaim double digestion after electrophoresis；With Ncol and Ndel
The rubella virus fusion protein gene fraction of double digestion chemical synthesis, electrophoresis reclaims the genetic fragment of double digestion, and -20 DEG C standby；
The genetic fragment of the plasmid fragments of double digestion and double digestion is pressed 1:3-10 ratios, with T4 ligases, 16 DEG C connect overnight, connection
Recombinant plasmid afterwards is PGEM-5zf-E1+EII+C；
(3) screening and identification of recombinant plasmid
By recombinant plasmid transformed e. coli bl21 (DE3), it is coated with the LB flat boards containing ampicillin, 37 DEG C of constant incubators
Overnight；Next day random picking conversion bacterium colony and control bacterium colony, extract plasmid, respectively with Ndel and Ncol double digestions, digestion respectively
After run electrophoresis, can see corresponding purpose fragment and carrier, construction of expression vector success, as positive expression bacterium.
7. a kind of preparation method of fusion protein for detecting Detecting Rubella Virus Antibodies In Human Sera according to claim 4, it is characterised in that
The preparation method also comprises the following steps：
Recombinant protein engineering bacteria high efficient expression：
Positive expression bacterium is seeded in the LB culture mediums containing ampicillin, 37 DEG C of constant-temperature tables vibrate 4h, plus final concentration of
0.5-1.5mmol/l IPTG, continue to induce 3-8h under the conditions of 37 DEG C, precipitation thalline are collected by centrifugation, precipitation thalline is broken
It is broken, take supernatant precipitation to carry out the destination protein expressed in SDS-PAGE detections, supernatant respectively；
The purifying of expressing protein：
Recombinant protein engineering bacteria is centrifuged into 8-12min under the conditions of 4 DEG C with 8000-12000rpm rotating speed, by the thalline of precipitation
In 1/10 lysate for being resuspended in former centrifugation volume, ice-bath ultrasonic thalline, with 10000-13000rpm rotating speed under the conditions of 4 DEG C
25-35min is centrifuged, bacterium solution supernatant is collected；Rinsed with equilibrium liquid after balance nickel ion affinity chromatograph post, the bacterium solution that ultrasound is collected
Pillar is washed with equilibrium liquid after the direct upper prop of supernatant, end of the sample, elution albumen is used successively, each eluting peak egg is collected
In vain, each peak albumen of SDS-PAGE electrophoresis detections.
8. a kind of preparation method of fusion protein for detecting Detecting Rubella Virus Antibodies In Human Sera according to claim 7, it is characterised in that:
Lysate in the purification step of expressing protein is made up of 50mM Tris-Hcl, 10mM EDTA, 15mM Nacl, 10mM DTT.
9. a kind of preparation method of fusion protein for detecting Detecting Rubella Virus Antibodies In Human Sera according to claim 7, it is characterised in that:
The imidazole elution that eluent in the purification step of expressing protein is concentration 20-500mM.
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