CN105820257B - Adenovirus hominis epitope chimeric protein and its preparation, application - Google Patents
Adenovirus hominis epitope chimeric protein and its preparation, application Download PDFInfo
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Abstract
The present inventor's Adenovirus Antigen epitope chimeric protein and its preparation, using being related to technique for gene engineering, vaccine and diagnostic reagent field.The amino acid sequence of adenovirus type III, 7 types, 11 types, 14 types and 55 type hexons is analyzed by computer, the protein fragments containing strong antigen are filtered out respectively, it is attached between segment with two glycine and a serine, form the concatenated chimeric protein of antigen fragment more than one, select the codon of protokaryon preference, it is transcribed into corresponding nucleic acid sequence, chemical synthesis full-length gene.Technique for gene engineering expression and purification obtains the chimeric protein, 363 amino acid of chimeric protein overall length.The chimeric protein of expression can be used for vaccine research, HAdV antibody or detection of antigen etc., and the present invention relates to technique for gene engineering, vaccine and diagnostic reagent fields.
Description
Technical field
The present inventor's Adenovirus Antigen epitope chimeric protein and its preparation, using being related to technique for gene engineering, vaccine and examine
Disconnected reagent field.The present invention is by 3 type of adenovirus hominis (HAdV), 7 types, 11 types, 14 types and 55 type specific antigen epitope eggs
The series connection of white tiles section, using technique for gene engineering, preparation is containing there are five types of the chimeric proteins of type adenovirus strong antigen epitope.Pass through meter
The analysis of calculation machine, filters out the protein fragments containing strong antigen epitope, protein fragments from five kinds of type adenovirus hexons
Between with two glycine and the connection of serine, be connected into epitope chimeric protein more than one.This is chimeric for chemical synthesis
The gene order of albumen, and the chimeric protein is expressed using technique for gene engineering.The chimeric protein of expression can be used for HAdV antibody
Or detection and the development of vaccine of antigen etc..
Background technique
Adenovirus hominis, i.e. HAdV(Human Adenovirus), it is that one kind has in Dense crowds such as kindergarten, armies
The virus of high harmfulness.Since in December, 2011, China different regions successively occur a lot of to break out biography through respiratory infectious
It catches an illness epidemic situation, epidemic situation involves wide, and infectiousness is strong, is respectively 55 type of B group, 7 types and 14 type glands through the identification of laboratory Pathogen test
Virus.
Adenovirus mainly causes respiratory disease, but can also infect the positions such as alimentary canal, the urinary tract, eye, cardiac muscle and draw
Play disease.It has been generally acknowledged that B1, C, E group adenovirus mainly cause respiratory disease, and B2 group mainly causes urinary system infection contamination.Entirely
Ball repeatedly reports the respiratory disease caused by adenovirus outbreak of epidemic in new recruit.
Adenovirus is a kind of uncanned double-stranded DNA virus, belongs to Adenoviridae, full-length genome 34.7kb, capsid is in rule
20 face body structures then, diameter 80-100nm.Core is made of distrand DNA and protein, there is nucleocapsid outside, above there is 252 particles,
It conjuncted is made of 240 hexons and 12 five.Epitope in hexon is to distinguish the standard of different serotypes, is virus to exempting from
The position that epidemic disease selects pressure most sensitive.
Currently, there are two types of the method for laboratory testing adenovirus infection is general.First is that special by polymerase chain reaction
Property detection adenoviral nucleic acid, the method is more complex, not easy to operate, and adenovirus parting is more difficult.Second is that utilizing Enzyme-linked Immunosorbent Assay
Specific IgM and IgG antibody in experiment detection serum.Under normal circumstances, when virus infection, human immune system can be excited
Virus is removed in humoral immunity and cell immune response and gradually control infection.When viremia virusemia occurs in infection early stage, from patient's blood
It can detecte viral nucleic acid in cleer and peaceful nose, pharyngeal secretion object.Stronger immune response can be induced after adenovirus infection, generated special
Property antibody.1 week after the onset of general, the IgM of patient's body starts to generate, and 7-10 days IgG start to generate, and then gradually rises.Cause
This is using the specific antibody in specific antigen detection serum.But a kind of antigen is typically only capable to detect a kind of type at present
Adenovirus antibody makes a definite diagnosis sense so needing to improve antibody detection rate using a variety of antigens when detecting HAdV antibody in serum
Type is contaminated, improves testing cost, and there are still certain probability of false negative.Developing multiple epitopes recombinant antigen is
The developing direction of adenovirus specific antibody quick detection reagent is established, and the developing direction to develop vaccine.
Summary of the invention
Object of the present invention is to provide a kind of adenovirus hominis epitope chimeric protein, adenovirus hominis in view of the above shortcomings
(HAdV) there are a variety of epitopes on the hexon of shell, be the virion position most sensitive to Immune Selection pressure, induction
Body generates a variety of specific antibodies, so the albumen is used as the reason that antigen develops adenovirus hominis (HAdV) antibody test reagent
Think albumen.
The amino acid sequence that adenovirus hexon is analyzed by computer, filters out five kinds of type adenovirus respectively and contains
There are the protein fragments of strong antigen epitope, is connected, be connected into more than one with two glycine and a serine between protein fragments
Epitope chimeric protein.The gene order of the chemical synthesis chimeric protein, and the chimeric egg is expressed using technique for gene engineering
It is white.The chimeric protein of expression can be used for the detection of HAdV antibody or antigen and the development of vaccine etc..
The present invention is antigen table of the chemical synthesis adenovirus hominis (HAdV) by adenovirus type III, 7 types, 11 types, 14 types and 55 types
The gene order of position chimeric protein prepares the chimeric egg containing five seed type Adenovirus Antigen epitopes using technique for gene engineering
It is white.The amino acid sequence that adenovirus hominis 3,7,11,14 and 55 type hexons are analyzed by computer is filtered out respectively containing strong
The protein fragments of epitope.Adenovirus type III filters out four protein fragments altogether, is the 136th~151 amino acids respectively, altogether
16 amino acid；164th~187 amino acids, totally 24 amino acid；265th~284 amino acids, totally 20 amino acid；The
422~437 amino acids, totally 16 amino acid.Adenovirus type 7 filters out four protein fragments altogether, is the 136th~148 respectively
Amino acids, totally 13 amino acid；161st~181 amino acids, totally 21 amino acid；258th~276 amino acids, totally 19
A amino acid；415th~426 amino acids, totally 12 amino acid.Adenovirus type XI totally two protein fragments, are the 136th respectively
~160 amino acids, totally 25 amino acid；174th~186 amino acids, totally 13 amino acid.14 type of adenovirus totally four eggs
White tiles section is the 136th~160 amino acids respectively, totally 25 amino acid；174th~196 amino acids, totally 23 amino acid；
268th~286 amino acids, totally 19 amino acid；415th~450 amino acids, totally 36 amino acid.55 type of adenovirus is total
Two protein fragments are the 136th~158 amino acids respectively, totally 23 amino acid；172nd~184 amino acids, totally 13
Amino acid.With two glycine and a serine connection between protein fragments, it is connected into epitope chimeric protein more than one,
363 amino acid of overall length.The codon for selecting prokaryotes preference, translates into corresponding nucleotide sequence for chimeric protein, changes
Learn synthesis full-length gene order.Utilize the technique for gene engineering expression and purification chimeric protein.The chimeric protein can be used for vaccine and
The detection of HAdV antibody or antigen, and for the immune anti-HAdV monoclonal antibody and how anti-etc. of preparing.
Adenovirus hominis (HAdV) chimeric protein and its preparation, using taking following steps to implement:
1. the screening of five seed type Adenovirus Antigen epitopes and the chemical synthesis of chimeric protein genetic fragment:
The amino of 3 type of adenovirus hominis, 7 types, 11 types, 14 types and 55 type hexons is analyzed using softwares such as ANTHEWIN
Acid sequence filters out the protein fragments in each type adenovirus hexon with strong antigen respectively.Between protein fragments
With two glycine and a serine connection, it is connected into epitope chimeric protein more than one.Select prokaryotes preference
Chimeric protein is translated into corresponding nucleotide sequence by codon.It is increased at the end 5' of chimeric protein genetic fragmentNdeI enzyme
Enzyme site (lower draw lines part), the end 3' increase terminator codon (TAA) andEcoRI restriction enzyme site (the lower part that draws lines).Make
The genetic fragment of synthesis is easy to be cloned into plasmid pET-28a (+)NdeI andEcoIn RI restriction enzyme site.This is embedding for chemical synthesis
The full-length gene order of hop protein.
The adenovirus type III epitope amino acid sequence of screening:
136th~151 aa:
164th~187 aa:
265th~284 aa:
422nd~437 aa:
The Adenovirus type 7 epitope amino acid sequence of screening:
136th~148 aa:
161st~181 aa:
258th~276 aa:
415th~426 aa:
The adenovirus type XI epitope amino acid sequence of screening:
136th~160 aa:
174th~186 aa:
The 14 type epitope amino acid sequence of adenovirus of screening:
136th~160 aa:
174th~196 aa:
268th~286 aa:
415th~450 aa:
The 55 type epitope amino acid sequence of adenovirus of screening:
136th~158 aa:
172nd~184 aa:
Adenovirus type III epitope amino acid sequence:
Ile Val Thr Thr Asn Gly Asp Asn Ala Val Thr Thr Thr Thr Asn Thr Gly
Gly Ser
Lys Glu Gly Leu Gln Ile Gly Lys Asp Ile Thr Thr Thr Glu Gly Glu Glu
Lys Pro
Ile Tyr Ala Asp Lys Gly Gly Ser Asp Gly Arg Asp Ala Val Ala Gly Ala
Leu Ala
Pro Glu Ile Val Leu Tyr Thr Glu Asn Gly Gly Ser Ile Lys Val Lys Thr
Asp Asp
Thr Asn Gly Trp Glu Lys Asp Ala Asn
Adenovirus type 7 epitope amino acid sequence:
Ile Val Thr Thr Gly Glu Asp Asn Ala Thr Thr Tyr Thr Gly Gly Ser Lys
Glu Gly
Leu Glu Ile Gly Lys Asp Ile Thr Ala Asp Asn Lys Pro Ile Tyr Ala Asp
Lys Gly
Gly Ser Asp Gly Arg Glu Ala Ala Asp Ala Phe Ser Pro Glu Ile Val Leu
Tyr Thr
Glu Asn Gly Gly Ser Ile Lys Pro Arg Asp Thr Ala Trp Glu Lys Asp Thr
Adenovirus type XI epitope amino acid sequence:
Ile Ala Glu Gly Val Lys Asn Thr Thr Gly Glu Glu His Val Thr Glu Glu
Glu Thr
Asn Thr Thr Thr Tyr Thr Gly Gly Ser Lys Glu Gly Leu Pro Val Gly Leu
Glu Val
Ser Asp Glu
14 type epitope amino acid sequence of adenovirus:
Leu Asp Lys Gly Val Glu Thr Thr Glu Glu Arg Gln Asn Glu Asp Gly Glu
Asn Asp
Glu Lys Ala Thr Tyr Thr Gly Gly Ser Lys Asp Gly Leu Pro Ile Gly Leu
Glu Val
Pro Ala Glu Gly Asp Pro Lys Pro Ile Tyr Ala Asn Lys Gly Gly Ser Asp
Leu Arg
Ser Gln Lys Gln Gly Leu Lys Pro Lys Ile Val Met Tyr Ala Glu Asn Gly
Gly Ser
Ile Gly Pro Arg Thr Asp Ser Tyr Lys Glu Ile Gln Leu Asn Gly Asp Gln
Ala Trp
Lys Asp Val Asn Pro Asn Gly Ile Ser Glu Leu Val Lys Gly Asn Pro Phe
55 type epitope amino acid sequence of adenovirus:
Ile Ala Glu Gly Val Lys Asn Gly Glu Glu Arg Val Thr Glu Glu Glu Asn
Asn Thr
Thr Thr Tyr Thr Gly Gly Ser Lys Glu Gly Leu Pro Ile Gly Leu Lys Val
Ser Asp
Glu
Chimeric protein amino acid sequence:
Ile Val Thr Thr Asn Gly Asp Asn Ala Val Thr Thr Thr Thr Asn Thr Gly
Gly Ser
Lys Glu Gly Leu Gln Ile Gly Lys Asp Ile Thr Thr Thr Glu Gly Glu Glu
Lys Pro
Ile Tyr Ala Asp Lys Gly Gly Ser Asp Gly Arg Asp Ala Val Ala Gly Ala
Leu Ala
Pro Glu Ile Val Leu Tyr Thr Glu Asn Gly Gly Ser Ile Lys Val Lys Thr
Asp Asp
Thr Asn Gly Trp Glu Lys Asp Ala Asn Gly Gly Ser Ile Val Thr Thr Gly
Glu Asp
Asn Ala Thr Thr Tyr Thr Gly Gly Ser Lys Glu Gly Leu Glu Ile Gly Lys
Asp Ile
Thr Ala Asp Asn Lys Pro Ile Tyr Ala Asp Lys Gly Gly Ser Asp Gly Arg
Glu Ala
Ala Asp Ala Phe Ser Pro Glu Ile Val Leu Tyr Thr Glu Asn Gly Gly Ser
Ile Lys
Pro Arg Asp Thr Ala Trp Glu Lys Asp Thr Gly Gly Ser Ile Ala Glu Gly
Val Lys
Asn Thr Thr Gly Glu Glu His Val Thr Glu Glu Glu Thr Asn Thr Thr Thr
Tyr Thr
Gly Gly Ser Lys Glu Gly Leu Pro Val Gly Leu Glu Val Ser Asp Glu Gly
Gly Ser
Leu Asp Lys Gly Val Glu Thr Thr Glu Glu Arg Gln Asn Glu Asp Gly Glu
Asn Asp
Glu Lys Ala Thr Tyr Thr Gly Gly Ser Lys Asp Gly Leu Pro Ile Gly Leu
Glu Val
Pro Ala Glu Gly Asp Pro Lys Pro Ile Tyr Ala Asn Lys Gly Gly Ser Asp
Leu Arg
Ser Gln Lys Gln Gly Leu Lys Pro Lys Ile Val Met Tyr Ala Glu Asn Gly
Gly Ser
Ile Gly Pro Arg Thr Asp Ser Tyr Lys Glu Ile Gln Leu Asn Gly Asp Gln
Ala Trp
Lys Asp Val Asn Pro Asn Gly Ile Ser Glu Leu Val Lys Gly Asn Pro Phe
Gly Gly
Ser Ile Ala Glu Gly Val Lys Asn Gly Glu Glu Arg Val Thr Glu Glu Glu
Asn Asn
Thr Thr Thr Tyr Thr Gly Gly Ser Lys Glu Gly Leu Pro Ile Gly Leu Lys
Val Ser
Asp Glu
The DNA sequence dna (1104bp) of chemically synthesized chimeric protein
2. expressing the building of HAdV epitope chimeric protein recombinant plasmid:
Extract plasmid pET-28a(+) (U.S.'s Novagen Products), it usesNdeI andEcoRI double digestion, after electrophoresis
The plasmid large fragment for recycling digestion, is dissolved in deionized water.WithNdeIWithEcoThe chemically synthesized HAdV antigen table of R I double digestion
Position chimeric protein genetic fragment is dissolved in deionized water after electrophoresis recycling.DNA fragmentation after above-mentioned digestion is taken, carrier is inserted into
PET-28a(+ in)NdeIWithEcoBetween R I site, the chimeric protein containing five seed type Adenovirus Antigen epitopes is expressed.
3. the screening and identification of recombinant plasmid:
By recombinant plasmid transformed e. coli bl21 (DE3), coating contains kanamycins (60 μ g/mL) LB plate, sets 37 DEG C
Overnight.Next day random picking conversion bacterium colony and control bacterium (plasmid pET-28a transformed bacteria), extract plasmid, bis- with NdeI and EcoRI
Digestion verification, 1.0% agarose gel electrophoresis results show to cut the target gene fragment of 1104bp.Meanwhile it will contain and be somebody's turn to do
The plasmid of genetic fragment carries out DNA sequence analysis, and sequence analysis confirms that recombinant plasmid contains the five seed type glands to link together
Virus antigen epitope genetic fragment, sequence are completely correct:
ATT GTT ACG ACG AAC GGT GAT AAT GCT GTT ACG ACG ACG ACG AAT ACG GGC
GGC TCC AAA GAA GGT CTG CAA ATC GGC AAA GAC ATC ACC ACG ACC GAA GGC GAA GAA
AAA CCG ATC TAC GCA GAT AAA GGC GGT AGT GAT GGT CGC GAT GCA GTT GCT GGT GCC
CTG GCA CCG GAA ATT GTC CTG TAT ACC GAA AAC GGC GGT AGC ATC AAA GTT AAA ACC
GAT GAC ACG AAC GGC TGG GAA AAA GAT GCG AAT GGC GGT AGC ATC GTC ACG ACC GGT
GAA GAC AAC GCC ACG ACC TAT ACC GGC GGT TCT AAA GAA GGC CTG GAA ATT GGT AAA
GAT ATC ACC GCA GAC AAT AAA CCG ATT TAC GCT GAT AAA GGC GGT AGC GAC GGT CGT
GAA GCG GCC GAT GCG TTT TCT CCG GAA ATT GTG CTG TAT ACC GAA AAT GGC GGT TCA
ATC AAA CCG CGC GAT ACC GCA TGG GAA AAA GAC ACG GGC GGT TCG ATT GCT GAA GGC
GTG AAA AAC ACG ACC GGT GAA GAA CAT GTT ACC GAA GAA GAA ACG AAT ACG ACC ACG
TAC ACC GGC GGT TCT AAA GAA GGC TTA CCG GTG GGT CTG GAA GTT AGT GAT GAA GGC
GGT TCC CTG GAC AAA GGC GTT GAA ACC ACG GAA GAA CGT CAG AAC GAA GAT GGT GAA
AAT GAC GAA AAA GCC ACC TAT ACG GGC GGT AGC AAA GAT GGT CTG CCG ATT GGC CTG
GAA GTG CCG GCC GAA GGC GAT CCG AAA CCG ATT TAT GCC AAC AAA GGC GGT AGC GAT
CTG CGT TCT CAG AAA CAA GGT CTG AAA CCG AAA ATT GTT ATG TAT GCA GAA AAC GGC
GGT TCA ATC GGC CCG CGC ACC GAT TCG TAC AAA GAA ATT CAG CTG AAT GGT GAT CAA
GCT TGG AAA GAC GTC AAC CCG AAT GGC ATC AGT GAA CTG GTG AAA GGT AAC CCG TTC
GGC GGT TCC ATT GCC GAA GGC GTC AAA AAT GGT GAA GAA CGC GTG ACC GAA GAA GAA
AAC AAT ACG ACG ACG TAC ACG GGC GGT AGC AAA GAA GGT CTG CCG ATT GGT CTG AAA
GTC TCC GAT GAA TAA
The expression of recombinant plasmid of building contains the chimeric protein of five seed type Adenovirus Antigen epitopes, 363 amino of overall length
Acid is adenovirus type III epitope protein segment in its N-terminal, is sequentially connected in series 7 types, 11 types, 14 type epitope protein segments, C
End is 55 type epitope protein segment of adenovirus, and each fragment length is followed successively by long 85 amino acid of 3 types, long 74 amino of 7 types
Acid, long 41 amino acid of 11 types, long 112 amino acid of 14 types, long 39 amino acid of 55 types, between segment by two glycine and
One serine connection, amino acid sequence are as follows:
Ile Val Thr Thr Asn Gly Asp Asn Ala Val Thr Thr Thr Thr Asn Thr Gly
Gly Ser
Lys Glu Gly Leu Gln Ile Gly Lys Asp Ile Thr Thr Thr Glu Gly Glu Glu
Lys Pro
Ile Tyr Ala Asp Lys Gly Gly Ser Asp Gly Arg Asp Ala Val Ala Gly Ala
Leu Ala
Pro Glu Ile Val Leu Tyr Thr Glu Asn Gly Gly Ser Ile Lys Val Lys Thr
Asp Asp
Thr Asn Gly Trp Glu Lys Asp Ala Asn Gly Gly Ser Ile Val Thr Thr Gly
Glu Asp
Asn Ala Thr Thr Tyr Thr Gly Gly Ser Lys Glu Gly Leu Glu Ile Gly Lys
Asp Ile
Thr Ala Asp Asn Lys Pro Ile Tyr Ala Asp Lys Gly Gly Ser Asp Gly Arg
Glu Ala
Ala Asp Ala Phe Ser Pro Glu Ile Val Leu Tyr Thr Glu Asn Gly Gly Ser
Ile Lys
Pro Arg Asp Thr Ala Trp Glu Lys Asp Thr Gly Gly Ser Ile Ala Glu Gly
Val Lys
Asn Thr Thr Gly Glu Glu His Val Thr Glu Glu Glu Thr Asn Thr Thr Thr
Tyr Thr
Gly Gly Ser Lys Glu Gly Leu Pro Val Gly Leu Glu Val Ser Asp Glu Gly
Gly Ser
Leu Asp Lys Gly Val Glu Thr Thr Glu Glu Arg Gln Asn Glu Asp Gly Glu
Asn Asp
Glu Lys Ala Thr Tyr Thr Gly Gly Ser Lys Asp Gly Leu Pro Ile Gly Leu
Glu Val
Pro Ala Glu Gly Asp Pro Lys Pro Ile Tyr Ala Asn Lys Gly Gly Ser Asp
Leu Arg
Ser Gln Lys Gln Gly Leu Lys Pro Lys Ile Val Met Tyr Ala Glu Asn Gly
Gly Ser
Ile Gly Pro Arg Thr Asp Ser Tyr Lys Glu Ile Gln Leu Asn Gly Asp Gln
Ala Trp
Lys Asp Val Asn Pro Asn Gly Ile Ser Glu Leu Val Lys Gly Asn Pro Phe
Gly Gly
Ser Ile Ala Glu Gly Val Lys Asn Gly Glu Glu Arg Val Thr Glu Glu Glu
Asn Asn
Thr Thr Thr Tyr Thr Gly Gly Ser Lys Glu Gly Leu Pro Ile Gly Leu Lys
Val Ser
Asp Glu
4. the screening and identification of expressed fusion protein engineering bacteria:
By the positive transformant containing recombinant plasmid, it is seeded to the culture medium of LB containing 3mL (the 60 μ g/mL containing kanamycin)
In test tube, 37 DEG C of shaken cultivation 3h add IPTG to final concentration 0.5mmol/L, continue shaken cultivation and induce 6h, bacterium is collected by centrifugation
Body carries out SDS-PAGE detection, and recon expresses relative molecular weight and is the HAdV chimeric protein of 55 kD, and compares bacterium BL21
(DE3) without this protein band.
5. the purifying of Adenovirus Antigen epitope chimeric protein:
1) the ultrasound cracking of Adenovirus Antigen epitope chimeric protein engineering bacteria
Bacterium is received into the engineering bacteria centrifugation (8000 rpm, 10 min, 4 DEG C) of inducing expression chimeric protein, thallus is resuspended in original
In the bacterial lysate (20 mmol/L PB pH7.4,5% glycerol) of 1/10 volume of culture solution, ice-bath ultrasonic breaks bacterium, and centrifugation is received
Collect supernatant.
2) the amine sulfate fractional precipitation of Adenovirus Antigen epitope chimeric protein
Supernatant volume accurately is measured, appropriate saturation sulfuric acid amine aqueous solution is added into supernatant, it is stirring while adding, make amine sulfate
Final concentration of 25%, it sets in ice bath overnight.Supernatant is collected by centrifugation, appropriate solid sulfur acid amide powder is added, it is stirring while adding, make sulphur
Final concentration of the 50% of acid amide is set in ice bath overnight.Precipitating is collected by centrifugation in next day.With 200mL equilibrium liquid (50mM Tris-HCl,
PH 7.5) suspend precipitating, and it is packed into bag filter, to 1000mL equilibrium liquid dialysed overnight, changes liquid 4 times.Supernatant is collected by centrifugation, directly
Upper DEAE-Separose FF anion column purifying.
3) DEAE-Separose FF anion column purifies
Balance DEAE-Separose FF anion column is rinsed with equilibrium liquid (50Mm Tris-HCl, pH 7.5), then will
The direct upper prop of the supernatant that upper step has been dialysed, about 100 mL/h of flow velocity.Column is sufficiently washed with equilibrium liquid after end of the sample, then successively with containing
100, the equilibrium liquid of 200,300,400,500,1000 mmol/L NaCl elutes albumen, each eluting peak albumen is collected, with 12%
Each peak albumen of SDS-PAGE electrophoresis detection determines Adenovirus Antigen epitope chimeric protein of which eluting peak containing expression.300
It is Adenovirus Antigen epitope chimeric protein that mmol/L NaCl, which elutes protein peak,.
6. by the Adenovirus Antigen epitope chimeric protein of expression, for vaccine, HAdV antibody or antigen detection and be used for
The immune anti-HAdV monoclonal antibody and how anti-etc. of preparing.
7. the strong antigen protein fragments of five kinds of type adenovirus hexons are connected, in the form of fusion protein into
Row expression, preparation.
8. by gene recombination technology, using yeast cells, insect cell, mammalian cell and genetically modified animals and plants into
Row recombinant expression prepares Adenovirus Antigen epitope chimeric protein.
English abbreviation explanation: HAdV (Human adenovirus): adenovirus hominis;EDTA: tetramethylethylenediamine；
IPTG: isopropylthiogalactoside；DTT: dithiothreitol (DTT)；SDS: dodecyl sodium sulfonate is received；PAGE: polyacrylamide
Gel electrophoresis；PB: phosphate buffer；KD: kilodalton.
The present invention has the advantage that compared with prior art
The Adenovirus Antigen epitope chimeric protein that we express, there is more advantages:
1. five kinds of type Adenovirus Antigen neoepitope Western segment compositions are expressed, than using individual protein fragments more respectively
It is convenient, economical.When making the antigen anti-HAdV antibody of detection with chimeric protein, it is no longer necessary to prepare these protein fragments respectively, also not
Need to adjust the use ratio of these antigens, therefore application is convenient and cost is relatively low.
2. presently used adenovirus vaccine is mainly inactivated vaccine and attenuated live vaccine, but adenovirus have to animal it is carcinogenic
Property, it is dangerous big, therefore the novel nucleocapsid Component Vaccines without DNA of exploitation seem extremely urgent.We utilize technique for gene engineering
The chimeric protein of expression preparation adenovirus hexon epitope containing there are many, lays the foundation to develop recombinant vaccine.Base
Because engineered vaccine have the advantages that safety, it is at low cost.
3. the chimeric protein containing five seed type Adenovirus Antigen epitopes of expression exists with soluble form, it is easy to pure
Change, and does not need renaturation process.
Detailed description of the invention
Below with reference to attached drawing, the invention will be further described:
Fig. 1 is the result that molecular biology software analyzes adenovirus type III hexon epitope.As a result it shows
Show, contain 4 strong hydrophily epitopes in adenovirus type III hexon N-terminal: 136~151aa, 164~187aa,
265~284aa, 422~437aa scheme the position of interior arrows.
Fig. 2 is the result that molecular biology software analyzes Adenovirus type 7 hexon epitope.As a result it shows
Show, contain 4 strong hydrophily epitopes: 136~148aa, 161~181aa in Adenovirus type 7 hexon N-terminal,
258~276aa, 415~426aa scheme the position of interior arrows.
Fig. 3 is the result that molecular biology software analyzes adenovirus type XI hexon epitope.As a result
Display contains 2 strong hydrophily epitopes in adenovirus type XI hexon N-terminal: 136~160aa, 174~
186aa schemes the position of interior arrows.
Fig. 4 is the result that molecular biology software analyzes 14 type hexon epitope of adenovirus.As a result
Display contains 4 strong hydrophily epitopes in 14 type hexon N-terminal of adenovirus: 136~160aa, 174~
196aa, 268~286aa, 415~450aa scheme the position of interior arrows.
Fig. 5 is the result that molecular biology software analyzes 55 type hexon epitope of adenovirus.As a result
It has been shown that, contains 2 strong hydrophily epitopes: 136~158aa, 172~184aa in adenovirus type III hexon N-terminal,
The position of arrows in scheming.
Fig. 6 is the construction of recombinant plasmid flow chart for expressing five kinds of type Adenovirus Antigen epitope chimeric proteins.
Fig. 7 is the SDS-PAGE analysis result for expressing Adenovirus Antigen epitope chimeric protein recombinant bacterium.By the recombination of building
Plasmid is transformed into Escherichia coli, chooses the SDS-PAGE electrophoresis of screening superior strain after single colonie.M: albumen marker；Yin:
Containing pET-28a(+) the negative control bacterium of plasmid；1-5: Adenovirus Antigen epitope chimeric protein recombinant bacterium, five equal tables of recon
The chimeric protein for being 55kDa up to relative molecular weight, i.e., the position of arrows in figure.
Fig. 8 is to express the SDS-PAGE analysis result of Adenovirus Antigen epitope chimeric protein after purification.M: albumen Marker；
1: Adenovirus Antigen epitope chimeric protein after purification, concentration 0.5mg/mL, relative molecular weight 55kDa；2: concentration is
The BSA of 0.5mg/mL.
Specific embodiment
The detailed description of embodiment of the present invention:
Analysis, gene chemical synthesis and the expression of adenovirus hexon epitope
The amino acid sequence of adenovirus type III, 7 types, 11 types, 14 types and 55 type hexons is analyzed by computer, point
Protein fragments wherein with strong antigen are not filtered out.Adenovirus type III filters out four protein fragments altogether, and Adenovirus type 7 is total
Filter out four protein fragments, adenovirus type XI totally two protein fragments, 14 type of adenovirus totally four protein fragments, adenovirus 55
Type totally two protein fragments.With two glycine and a serine connection between protein fragments, it is connected into antigen table more than one
Position chimeric protein.The codon for selecting prokaryotes preference, translates into corresponding nucleotide sequence for chimeric protein.Chemical synthesis
The full-length gene order of the chimeric protein, and be cloned into plasmid pET-28a (+)NdeI/EcoGland is expressed in the site RI
Virus antigen epitope chimeric protein.By recombinant plasmid transformed e. coli bl21 (DE3), screening obtains high efficient expression adenovirus
The engineering bacteria of epitope chimeric protein, the HAdV chimeric protein of expression account for the 60% of mycoprotein total amount.
Materials and methods
1. strain and plasmid: host strain BL21 (DE3) and expression vector pET-28a (+) is that Novagen company in the U.S. produces
Product.
2. molecular biology reagents: restriction enzymeNdeI、EcoRI and T4 DNA ligase is the production of TaKaRa company
Product.Plasmid purification kit and the kit that DNA fragmentation is recycled out of Ago-Gel are TaKaRa Products.DTT and
IPTG is TaKaRa Products.Other reagents are import or domestic analytical reagents.
3. the synthesis of genetic fragment: helping to synthesize by Dalian TaKaRa company.
4. gene clone method: the digestion of DNA, connection, electrophoresis；Extraction, the conversion of plasmid；The SDS-PAGE of albumen points
The general molecular cloning method such as analysis carries out according to a conventional method.Other kit by specifications are operated.
5. DNA sequence analysis: using TaKaRa company plasmid purification kit plasmid purification, surveyed with the full-automatic sequenator of DNA
Sequence.
As a result
The screening of 1.HAdV hexon epitope and its chemical synthesis of genetic fragment:
Using softwares such as ANTHEWIN, adenovirus type III, 7 types, 11 types, 14 types and 55 type hexons are analyzed by computer
The amino acid sequence of albumen, discovery have multiple segments to contain stronger antigenic determinant.Adenovirus type III filters out four eggs altogether
White tiles section (Fig. 1) is the 136th~151 aa respectively, totally 16 amino acid；164th~187 amino acids, totally 24 amino
Acid；265th~284 amino acids, totally 20 amino acid；422nd~437 amino acids, totally 16 amino acid.Adenovirus type 7
Four protein fragments (Fig. 2) are filtered out altogether, are the 136th~148 amino acids respectively, totally 13 amino acid；161st~181
Amino acid, totally 21 amino acid；258th~276 amino acids, totally 19 amino acid；415th~426 amino acids, totally 12
Amino acid.Adenovirus type XI totally two protein fragments (Fig. 3), are the 136th~160 amino acids respectively, totally 25 amino acid；The
174~186 amino acids, totally 13 amino acid.14 type of adenovirus totally four protein fragments (Fig. 4), are the 136th~160 respectively
Amino acids, totally 25 amino acid；174th~196 amino acids, totally 23 amino acid；268th~286 amino acids, totally 19
A amino acid；415th~450 amino acids, totally 36 amino acid.55 type of adenovirus totally two protein fragments (Fig. 5) are respectively
136th~158 amino acids, totally 23 amino acid；172nd~184 amino acids, totally 13 amino acid.Between protein fragments
With two glycine and a serine connection, it is connected into epitope chimeric protein more than one.Select prokaryotes preference
Chimeric protein is translated into corresponding nucleotide sequence by codon.It is increased at the end 5' of chimeric protein gene orderNdeI enzyme
Enzyme site, the end 3' increase terminator codon (TAA) andEcoRI restriction enzyme site.The overall length base of the chemical synthesis chimeric protein
Because of sequence.
The adenovirus type III epitope amino acid sequence of screening:
136th~151 aa:
Ile Val Thr Thr Asn Gly Asp Asn Ala Val Thr Thr Thr Thr Asn Thr
164th~187 aa:
Lys Glu Gly Leu Gln Ile Gly Lys Asp Ile Thr Thr Thr Glu Gly Glu Glu
Lys Pro
Ile Tyr Ala Asp Lys
265th~284 aa:
Asp Gly Arg Asp Ala Val Ala Gly Ala Leu Ala Pro Glu Ile Val Leu Tyr
Thr Glu
Asn
422nd~437 aa:
Ile Lys Val Lys Thr Asp Asp Thr Asn Gly Trp Glu Lys Asp Ala Asn
The Adenovirus type 7 epitope amino acid sequence of screening:
136th~148 aa:
Ile Val Thr Thr Gly Glu Asp Asn Ala Thr Thr Tyr Thr
161st~181 aa:
Lys Glu Gly Leu Glu Ile Gly Lys Asp Ile Thr Ala Asp Asn Lys Pro Ile
Tyr AlaAsp Lys
258th~276 aa:
Asp Gly Arg Glu Ala Ala Asp Ala Phe Ser Pro Glu Ile Val Leu Tyr Thr
Glu Asn
415th~426 aa:
Ile Lys Pro Arg Asp Thr Ala Trp Glu Lys Asp Thr
The adenovirus type XI epitope amino acid sequence of screening:
136th~160 aa:
Ile Ala Glu Gly Val Lys Asn Thr Thr Gly Glu Glu His Val Thr Glu Glu
Glu Thr
Asn Thr Thr Thr Tyr Thr
174th~186 aa:
Lys Glu Gly Leu Pro Val Gly Leu Glu Val Ser Asp Glu
The 14 type epitope amino acid sequence of adenovirus of screening:
136th~160 aa:
Leu Asp Lys Gly Val Glu Thr Thr Glu Glu Arg Gln Asn Glu Asp Gly Glu
Asn Asp
Glu Lys Ala Thr Tyr Thr
174th~196 aa:
Lys Asp Gly Leu Pro Ile Gly Leu Glu Val Pro Ala Glu Gly Asp Pro Lys
Pro Ile
Tyr Ala Asn Lys
268th~286 aa:
Asp Leu Arg Ser Gln Lys Gln Gly Leu Lys Pro Lys Ile Val Met Tyr Ala
Glu Asn
415th~450 aa:
Ile Gly Pro Arg Thr Asp Ser Tyr Lys Glu Ile Gln Leu Asn Gly Asp Gln
Ala Trp
Lys Asp Val Asn Pro Asn Gly Ile Ser Glu Leu Val Lys Gly Asn Pro Phe
The 55 type epitope amino acid sequence of adenovirus of screening:
136th~158 aa:
Ile Ala Glu Gly Val Lys Asn Gly Glu Glu Arg Val Thr Glu Glu Glu Asn
Asn Thr
Thr Thr Tyr Thr
172nd~184 aa:
Lys Glu Gly Leu Pro Ile Gly Leu Lys Val Ser Asp Glu
Adenovirus type III epitope amino acid sequence:
Ile Val Thr Thr Asn Gly Asp Asn Ala Val Thr Thr Thr Thr Asn Thr Gly
Gly Ser
Lys Glu Gly Leu Gln Ile Gly Lys Asp Ile Thr Thr Thr Glu Gly Glu Glu
Lys Pro
Ile Tyr Ala Asp Lys Gly Gly Ser Asp Gly Arg Asp Ala Val Ala Gly Ala
Leu Ala
Pro Glu Ile Val Leu Tyr Thr Glu Asn Gly Gly Ser Ile Lys Val Lys Thr
Asp Asp
Thr Asn Gly Trp Glu Lys Asp Ala Asn
Adenovirus type 7 epitope amino acid sequence:
Ile Val Thr Thr Gly Glu Asp Asn Ala Thr Thr Tyr Thr Gly Gly Ser Lys
Glu Gly
Leu Glu Ile Gly Lys Asp Ile Thr Ala Asp Asn Lys Pro Ile Tyr Ala Asp
Lys Gly
Gly Ser Asp Gly Arg Glu Ala Ala Asp Ala Phe Ser Pro Glu Ile Val Leu
Tyr Thr
Glu Asn Gly Gly Ser Ile Lys Pro Arg Asp Thr Ala Trp Glu Lys Asp Thr
Adenovirus type XI epitope amino acid sequence:
Ile Ala Glu Gly Val Lys Asn Thr Thr Gly Glu Glu His Val Thr Glu Glu
Glu Thr
Asn Thr Thr Thr Tyr Thr Gly Gly Ser Lys Glu Gly Leu Pro Val Gly Leu
Glu Val
Ser Asp Glu
14 type epitope amino acid sequence of adenovirus:
Leu Asp Lys Gly Val Glu Thr Thr Glu Glu Arg Gln Asn Glu Asp Gly Glu
Asn Asp
Glu Lys Ala Thr Tyr Thr Gly Gly Ser Lys Asp Gly Leu Pro Ile Gly Leu
Glu Val
Pro Ala Glu Gly Asp Pro Lys Pro Ile Tyr Ala Asn Lys Gly Gly Ser Asp
Leu Arg
Ser Gln Lys Gln Gly Leu Lys Pro Lys Ile Val Met Tyr Ala Glu Asn Gly
Gly Ser
Ile Gly Pro Arg Thr Asp Ser Tyr Lys Glu Ile Gln Leu Asn Gly Asp Gln
Ala Trp
Lys Asp Val Asn Pro Asn Gly Ile Ser Glu Leu Val Lys Gly Asn Pro Phe
55 type epitope amino acid sequence of adenovirus:
Ile Ala Glu Gly Val Lys Asn Gly Glu Glu Arg Val Thr Glu Glu Glu Asn
Asn Thr
Thr Thr Tyr Thr Gly Gly Ser Lys Glu Gly Leu Pro Ile Gly Leu Lys Val
Ser Asp
Glu
Chimeric protein amino acid sequence:
Ile Val Thr Thr Asn Gly Asp Asn Ala Val Thr Thr Thr Thr Asn Thr Gly
Gly Ser
Lys Glu Gly Leu Gln Ile Gly Lys Asp Ile Thr Thr Thr Glu Gly Glu Glu
Lys Pro
Ile Tyr Ala Asp Lys Gly Gly Ser Asp Gly Arg Asp Ala Val Ala Gly Ala
Leu Ala
Pro Glu Ile Val Leu Tyr Thr Glu Asn Gly Gly Ser Ile Lys Val Lys Thr
Asp Asp
Thr Asn Gly Trp Glu Lys Asp Ala Asn Gly Gly Ser Ile Val Thr Thr Gly
Glu Asp
Asn Ala Thr Thr Tyr Thr Gly Gly Ser Lys Glu Gly Leu Glu Ile Gly Lys
Asp Ile
Thr Ala Asp Asn Lys Pro Ile Tyr Ala Asp Lys Gly Gly Ser Asp Gly Arg
Glu Ala
Ala Asp Ala Phe Ser Pro Glu Ile Val Leu Tyr Thr Glu Asn Gly Gly Ser
Ile Lys
Pro Arg Asp Thr Ala Trp Glu Lys Asp Thr Gly Gly Ser Ile Ala Glu Gly
Val Lys
Asn Thr Thr Gly Glu Glu His Val Thr Glu Glu Glu Thr Asn Thr Thr Thr
Tyr Thr
Gly Gly Ser Lys Glu Gly Leu Pro Val Gly Leu Glu Val Ser Asp Glu Gly
Gly Ser
Leu Asp Lys Gly Val Glu Thr Thr Glu Glu Arg Gln Asn Glu Asp Gly Glu
Asn Asp
Glu Lys Ala Thr Tyr Thr Gly Gly Ser Lys Asp Gly Leu Pro Ile Gly Leu
Glu Val
Pro Ala Glu Gly Asp Pro Lys Pro Ile Tyr Ala Asn Lys Gly Gly Ser Asp
Leu Arg
Ser Gln Lys Gln Gly Leu Lys Pro Lys Ile Val Met Tyr Ala Glu Asn Gly
Gly Ser
Ile Gly Pro Arg Thr Asp Ser Tyr Lys Glu Ile Gln Leu Asn Gly Asp Gln
Ala Trp
Lys Asp Val Asn Pro Asn Gly Ile Ser Glu Leu Val Lys Gly Asn Pro Phe
Gly Gly
Ser Ile Ala Glu Gly Val Lys Asn Gly Glu Glu Arg Val Thr Glu Glu Glu
Asn Asn
Thr Thr Thr Tyr Thr Gly Gly Ser Lys Glu Gly Leu Pro Ile Gly Leu Lys
Val Ser
Asp Glu
The DNA sequence dna (1104bp) of chemically synthesized chimeric protein
2. the building of HAdV epitope chimeric protein recombinant plasmid:
Extract plasmid pET28a(+) (U.S.'s Novagen Products), it usesNdeI andEcoRI double digestion returns after electrophoresis
The plasmid large fragment for receiving digestion, is dissolved in deionized water.WithNdeIWithEcoThe chemically synthesized HAdV epitope of R I double digestion
Chimeric protein genetic fragment is dissolved in deionized water after electrophoresis recycling.DNA fragmentation after above-mentioned digestion is taken, carrier is inserted into
PET28a(+ in)NdeIWithEcoBetween R I site, expression is containing there are five types of the chimeric proteins of type Adenovirus Antigen epitope.
Building process is shown in Fig. 6.
3. the screening and identification of recombinant plasmid:
Converted product coating is contained kanamycins (60 by the recombinant plasmid transformed e. coli bl21 (DE3) that upper step is connected
μ g/mL) LB plate, set 37 DEG C overnight.Next day selects 5 conversion daughter colonys at random, is inoculated into cultivates containing 4 mL liquid LB respectively
In the test tube of base (the 60 μ g/mL containing kanamycins), 37 DEG C of shaken cultivation 6h are set, take bacterium solution 1mL, bacterium is received in centrifugation.Respectively with 50 μ L
Deionized water suspension thalline, boiling water boiling 5min are centrifuged (4 DEG C, 12000rpm) 5min, 1 μ L of supernatant (inside having plasmid) are taken to be used as
Pcr template, with chimeric protein genetic fragment 5 ' hold primer (5 '-CATATGATTGTTACGACGAACGGTGATAAT-3 ') and
Primer (the 5 '-GAATTCTTATT at 3 ' ends
CATCGGAGACTTT-3 ') composition primer pair, the positive recombinant plasmid of PCR amplification genetic fragment containing chimeric protein,
The genetic fragment of long 1104bp should be amplified.PCR reaction density are as follows: each 1 μ L of 1 μ L of plasmid template, upstream and downstream primer, 10x
5.0 μ L of Buffer, 2.5 mmol/L dNTP, 4.0 μ L, 0.5 μ L of Taq DNA polymerase (2.5 U), 37.5 μ of deionized water
L, 50 μ L of total volume.Amplification condition are as follows: 94 DEG C 30 seconds, 60 DEG C 30 seconds, 72 DEG C 60 seconds, 30 circulation；Last 72 DEG C extend 7
Minute.5 μ L of pcr amplification product is taken, with 1.2% Agarose gel detection, as a result, 8 transformants amplify 1104bp
Target gene fragment.It confirms, containing there are five types of the tandem genes of type Adenovirus Antigen epitope gene segment.
The plasmid of transformant is extracted, the tandem gene of five kinds of type Adenovirus Antigen epitope gene segments in plasmid is measured
Sequence, DNA sequence analysis confirm that recombinant plasmid contains there are five types of type Adenovirus Antigen epitope gene segment, and sequence is completely correct:
The expression of recombinant plasmid of building contains the chimeric protein there are five types of type Adenovirus Antigen epitope, 363 amino of overall length
Acid is adenovirus type III epitope protein segment in its N-terminal, is sequentially connected in series 7 types, 11 types, 14 type epitope protein segments, C
End is 55 type epitope protein segment of adenovirus, and each fragment length is followed successively by long 85 amino acid of 3 types, long 74 amino of 7 types
Acid, long 41 amino acid of 11 types, long 112 amino acid of 14 types, long 39 amino acid of 55 types, between segment by two glycine and
One serine connection, amino acid sequence are as follows:
Ile Val Thr Thr Asn Gly Asp Asn Ala Val Thr Thr Thr Thr Asn Thr Gly
Gly Ser
Lys Glu Gly Leu Gln Ile Gly Lys Asp Ile Thr Thr Thr Glu Gly Glu Glu
Lys Pro
Ile Tyr Ala Asp Lys Gly Gly Ser Asp Gly Arg Asp Ala Val Ala Gly Ala
Leu Ala
Pro Glu Ile Val Leu Tyr Thr Glu Asn Gly Gly Ser Ile Lys Val Lys Thr
Asp Asp
Thr Asn Gly Trp Glu Lys Asp Ala Asn Gly Gly Ser Ile Val Thr Thr Gly
Glu Asp
Asn Ala Thr Thr Tyr Thr Gly Gly Ser Lys Glu Gly Leu Glu Ile Gly Lys
Asp Ile
Thr Ala Asp Asn Lys Pro Ile Tyr Ala Asp Lys Gly Gly Ser Asp Gly Arg
Glu Ala
Ala Asp Ala Phe Ser Pro Glu Ile Val Leu Tyr Thr Glu Asn Gly Gly Ser
Ile Lys
Pro Arg Asp Thr Ala Trp Glu Lys Asp Thr Gly Gly Ser Ile Ala Glu Gly
Val Lys
Asn Thr Thr Gly Glu Glu His Val Thr Glu Glu Glu Thr Asn Thr Thr Thr
Tyr Thr
Gly Gly Ser Lys Glu Gly Leu Pro Val Gly Leu Glu Val Ser Asp Glu Gly
Gly Ser
Leu Asp Lys Gly Val Glu Thr Thr Glu Glu Arg Gln Asn Glu Asp Gly Glu
Asn Asp
Glu Lys Ala Thr Tyr Thr Gly Gly Ser Lys Asp Gly Leu Pro Ile Gly Leu
Glu Val
Pro Ala Glu Gly Asp Pro Lys Pro Ile Tyr Ala Asn Lys Gly Gly Ser Asp
Leu Arg
Ser Gln Lys Gln Gly Leu Lys Pro Lys Ile Val Met Tyr Ala Glu Asn Gly
Gly Ser
Ile Gly Pro Arg Thr Asp Ser Tyr Lys Glu Ile Gln Leu Asn Gly Asp Gln
Ala Trp
Lys Asp Val Asn Pro Asn Gly Ile Ser Glu Leu Val Lys Gly Asn Pro Phe
Gly Gly
Ser Ile Ala Glu Gly Val Lys Asn Gly Glu Glu Arg Val Thr Glu Glu Glu
Asn Asn
Thr Thr Thr Tyr Thr Gly Gly Ser Lys Glu Gly Leu Pro Ile Gly Leu Lys
Val Ser
Asp Glu
4. the screening and identification of expressed fusion protein engineering bacteria:
Above-mentioned transformant is seeded in the test tube of the culture medium of LB containing 3mL (the 60 μ g/mL containing kanamycin), 37 DEG C of oscillations
3h is cultivated, IPTG to final concentration 0.5mmol/L is added, continues shaken cultivation and induces 6h, thalline were collected by centrifugation carries out SDS-PAGE inspection
It surveys, the Adenovirus Antigen epitope chimeric protein (Fig. 7) that 5 transformant expression relative molecular weights are 55 kD.
The purifying of Adenovirus Antigen epitope chimeric protein
According to the amino acid sequence of Adenovirus Antigen epitope chimeric protein, its physicochemical property is analyzed, determines purifying appropriate
Method.Analyzed through computer, the isoelectric point of Adenovirus Antigen epitope chimeric protein is pH4.365, therefore we determine in pH be
In 7.5 50Mm Tris-HCl buffer, purified with DEAE-Separose FF anion column.Specific step is as follows:
Material and method
1. main agents:
DEAE-Separose FF polyanionic gel is Pharmacia Products, and IPTG, DTT are the production of TaTaRa company
Product.Other reagents are domestic or Import Analysis pure reagent.
2. the inducing expression and ultrasound cracking of Adenovirus Antigen epitope chimeric protein engineering bacteria
From the LB plate for being inoculated with No. 1 engineering bacteria, single colonie is chosen with toothpick, is inoculated into LB liquid medium containing 200mL
Conical flask in, add kanamycins to 60 μ g/mL of final concentration, set overnight incubation in 37 DEG C of shaking tables.Bacterium solution is inoculated by next day
The conical flask of 4 LB liquid mediums containing 200mL respectively, every bottle of inoculation bacterium solution 50mL set shaken cultivation 1 in 37 DEG C of shaking tables
Hour, then plus IPTG to final concentration 0.1mmol/L, inducing expression 4 hours.
Bacterium, thallus weight are received into the 1000mL engineering bacteria centrifugation (8000 rpm, 30 min, 4 DEG C) of inducing expression chimeric protein
It is suspended from the bacterial lysate (20 mmol/L PB pH7.4,10 mmol/L EDTA, 1 mmol/L DTT, 5% glycerol) of 100mL
Interior, ice-bath ultrasonic breaks 10 min of bacterium, and supernatant is collected in centrifugation (8000 rpm, 30 min, 4 DEG C).
3. the amine sulfate fractional precipitation of Adenovirus Antigen epitope chimeric protein
Supernatant volume accurately is measured, appropriate saturation sulfuric acid amine aqueous solution is added into supernatant, it is stirring while adding, make amine sulfate
Final concentration of 25%, it sets in ice bath overnight.Next day is centrifuged (12000 rpm, 20 min, 4 DEG C) and collects supernatant, and appropriate solid is added
Amine sulfate powder, it is stirring while adding, make final concentration of the 50% of amine sulfate, sets in ice bath overnight.Next day centrifugation (12000 rpm,
20 min, 4 DEG C) collect precipitating.It is suspended and is precipitated with 200mL equilibrium liquid (50mM Tris-HCl, pH 7.5), is packed into bag filter,
To 1000mL equilibrium liquid dialysed overnight, change liquid 4 times.It is centrifuged (12000 rpm, 20 min, 4 DEG C) and collects supernatant, directly upper DEAE-
The purifying of Separose FF anion column.
The purifying of 4.DEAE-Separose FF anion column
Balance DEAE-Separose FF anion column is rinsed with equilibrium liquid (50mM Tris-HCl, pH 7.5), then will
The direct upper prop of the supernatant that upper step has been dialysed, 100 mL/h of loading flow velocity.Column is sufficiently washed with equilibrium liquid after end of the sample, then successively
Albumen is eluted with the solution containing 100,200,300,400,500,1000 mmol/L NaCl, collects each eluting peak albumen, is used
Each peak albumen of 12% SDS-PAGE electrophoresis detection determines Adenovirus Antigen epitope chimeric protein of which eluting peak containing expression.
As a result
The albumen that various concentration NaCl is eluted from DEAE-Separose FF column carries out SDS-PAGE analysis, by electrophoresis
As a result it is found that Adenovirus Antigen epitope chimeric protein is present in 300 mmol/L NaCl elution protein peak.
By 300 mmol/L NaCl elution protein peak by dialysis concentration, SDS-PAGE measures chimeric protein concentration,
Know that the resulting chimeric protein concentration of purifying is 0.5mg/mL(Fig. 8).
The identification and application of Adenovirus Antigen epitope chimeric protein
The Adenovirus Antigen epitope chimeric protein of purifying is used as immunogene, BALB/c mouse is immunized, prepares antiserum.It is logical
Indirect ELISA experimental method is crossed, chimeric protein antiserum is detected using five kinds of type Adenovirus Antigen neoepitope Westerns, with
Identify the immunogenicity of Adenovirus Antigen epitope chimeric protein.Experimental result shows that the chimeric protein has good immunogene
Property, it can be used as the research of adenovirus vaccine.
Material and method
1. adenovirus type III, 7 types, 11 types, 14 types and 55 types epitope protein: by this laboratory prepare save.
2. integrated enzyme reaction material: elisa plate is that Shenzhen produces 96 orifice plates, the sheep anti mouse of horseradish peroxidase (HRP) label
IgG is purchased from Sigma company.Other materials are the conventional material of integrated enzyme reaction.
3. prepared by chimeric protein antiserum: after chimeric protein and Freund's complete adjuvant are sufficiently mixed emulsification in equal volume, abdomen
Chamber injecting immune mouse.Later every two weeks, by after chimeric protein and the isometric mixing and emulsifying of incomplete Freund's adjuvant, added
It is strong immune.It is immunized 4 times altogether.Two weeks after the 4th is immune, eye socket takes blood, prepares antiserum.
4. chimeric protein immunogenicity detects: being detected using indirect elisa method to chimeric protein immunogenicity.Base
This step are as follows: dilute five kinds of type Adenovirus Antigen neoepitope Westerns respectively with CBS buffer (pH9.6) to final concentration of 2 μ g/
ML is coated with 96 hole elisa Plates, every 100 μ L of hole, 4 DEG C of overnight incubations；Later with the abundant board-washing of 1 ‰ PBST 5 times, 10% small ox blood
(FBS) is used as confining liquid clearly, and every hole adds 150 μ L, 37 DEG C of closing 2h；The abundant board-washing of 1 ‰ PBST 5 times, chimeric protein antiserum
In following ratio gradient dilution (calf serum that dilution is 10%) 1:10000,1:20000,1:40000,1:80000,1:
160000,1:320000,1:640000,1:1280000,100 hole μ L/, 37 DEG C of incubation 2h, negative serum are diluted by 1:10000
As control；The abundant board-washing of 1 ‰ PBST 5 times；Add the sheep anti-mouse igg of 1:5000 dilution (calf serum that dilution is 10%),
100 holes μ L/, 37 DEG C of incubation 1h；The abundant board-washing of 1 ‰ PBST 5 times, is added TMB developing solution, and 50 μ L/hole, are protected from light by 37 DEG C
10min；The HCl of 1mol/L is added, 50 holes μ L/ terminate reaction；Microplate reader measures each hole A450Value, if P/N > 2.1, can sentence
Determine the result positive.
As a result
1. indirect elisa method detects Adenovirus Antigen epitope chimeric protein immunogenicity
The adenovirus type III of purifying, 7 types, 11 types, 14 types and 55 type epitope proteins are diluted with CBS buffer and are coated with
The antiserum that mouse obtains is immunized with Adenovirus Antigen epitope chimeric protein for ELISA Plate, detection.The results show that adenovirus type III egg
White detection chimeric protein serum antibody titer is 1:320000；Adenovirus type 7 Protein Detection chimeric protein serum antibody titer is
1:640000；Adenovirus type XI Protein Detection chimeric protein serum antibody titer is 1:640000；14 type Protein Detection of adenovirus
Chimeric protein serum antibody titer is 1:1280000；55 type Protein Detection chimeric protein serum antibody titer of adenovirus is 1:
640000.After illustrating that body is immunized in Adenovirus Antigen epitope chimeric protein, body can effectively be stimulated to generate for each type
The antibody of Adenovirus Antigen epitope has preferable immunogenicity, lays the foundation further to develop adenovirus vaccine.
Adenovirus hominis (HAdV) epitope chimeric protein sequence table sees appendix document: nucleotide or amino acid sequence meter
The readable carrier of calculation machine.
Adenovirus hominis (HAdV) epitope chimeric protein sequence table
<110>Li Yuexi
<120>adenovirus hominis epitope chimeric protein and its preparation, application
<160> 2
<210> 1
<211> 363
<212> PRT
<213>adenovirus hominis
<220>
<223>contain a plurality of types of adenovirus hominis epitopes.
<400> 1
<210> 2
<211> 1092
<212> DNA
<213>artificial sequence
<220>
<221> CDS
<222> (1)...(1092)
<223>artificial synthesized genetic fragment encodes the chimeric protein containing Adenovirus Antigen epitope.
<220>
<221> mis-feature
<222> (1090)...(1092)
<223>increased terminator codon when gene is synthesized.
<400> 2
Claims (4)
1. a kind of adenovirus hominis epitope chimeric protein, by adenovirus type III, 7 types, 11 types, 14 types and 55 type epitope eggs
White tiles section is formed by connecting, and is connected with glycine with serine between protein fragments, 363 amino acid of chimeric protein overall length, amino
Acid sequence is as follows:
。
2. a kind of encoding gene of adenovirus hominis epitope chimeric protein described in claim 1, it is characterised in that: its nucleosides
Acid sequence is as follows:
ATT GTT ACG ACG AAC GGT GAT AAT GCT GTT ACG ACG ACG ACG AAT ACG
GGC GGC TCC AAA GAA GGT CTG CAA ATC GGC AAA GAC ATC ACC ACG ACC
GAA GGC GAA GAA AAA CCG ATC TAC GCA GAT AAA GGC GGT AGT GAT GGT
CGC GAT GCA GTT GCT GGT GCC CTG GCA CCG GAA ATT GTC CTG TAT ACC
GAA AAC GGC GGT AGC ATC AAA GTT AAA ACC GAT GAC ACG AAC GGC TGG
GAA AAA GAT GCG AAT GGC GGT AGC ATC GTC ACG ACC GGT GAA GAC AAC
GCC ACG ACC TAT ACC GGC GGT TCT AAA GAA GGC CTG GAA ATT GGT AAA
GAT ATC ACC GCA GAC AAT AAA CCG ATT TAC GCT GAT AAA GGC GGT AGC
GAC GGT CGT GAA GCG GCC GAT GCG TTT TCT CCG GAA ATT GTG CTG TAT
ACC GAA AAT GGC GGT TCA ATC AAA CCG CGC GAT ACC GCA TGG GAA AAA
GAC ACG GGC GGT TCG ATT GCT GAA GGC GTG AAA AAC ACG ACC GGT GAA
GAA CAT GTT ACC GAA GAA GAA ACG AAT ACG ACC ACG TAC ACC GGC GGT
TCT AAA GAA GGC TTA CCG GTG GGT CTG GAA GTT AGT GAT GAA GGC GGT
TCC CTG GAC AAA GGC GTT GAA ACC ACG GAA GAA CGT CAG AAC GAA GAT
GGT GAA AAT GAC GAA AAA GCC ACC TAT ACG GGC GGT AGC AAA GAT GGT
CTG CCG ATT GGC CTG GAA GTG CCG GCC GAA GGC GAT CCG AAA CCG ATT
TAT GCC AAC AAA GGC GGT AGC GAT CTG CGT TCT CAG AAA CAA GGT CTG
AAA CCG AAA ATT GTT ATG TAT GCA GAA AAC GGC GGT TCA ATC GGC CCG
CGC ACC GAT TCG TAC AAA GAA ATT CAG CTG AAT GGT GAT CAA GCT TGG
AAA GAC GTC AAC CCG AAT GGC ATC AGT GAA CTG GTG AAA GGT AAC CCG
TTC GGC GGT TCC ATT GCC GAA GGC GTC AAA AAT GGT GAA GAA CGC GTG
ACC GAA GAA GAA AAC AAT ACG ACG ACG TAC ACG GGC GGT AGC AAA GAA
GGT CTG CCG ATT GGT CTG AAA GTC TCC GAT GAA。
3. a kind of method for preparing adenovirus hominis epitope chimeric protein as described in claim 1, which is characterized in that utilize
Technique for gene engineering expression prepares the albumen, the specific steps are as follows:
The screening of Adenovirus Antigen neoepitope Western segment:
Using softwares such as ANTHEWIN, adenovirus type III, 7 types, 11 types, 14 types and 55 type hexons are analyzed by computer
Amino acid sequence, discovery there are multiple segments to contain stronger antigenic determinant, adenovirus type III filters out four albumen flakes altogether
Section, is the 136th amino acids to the 151st amino acids respectively, totally 16 amino acid；164th amino acids to the 187th bit amino
Acid, totally 24 amino acid；265th amino acids to the 284th amino acids, totally 20 amino acid；422nd amino acids are to
437 amino acids, totally 16 amino acid, Adenovirus type 7 filter out four protein fragments altogether, be respectively the 136th amino acids extremely
148th amino acids, totally 13 amino acid；161st amino acids to the 181st amino acids, totally 21 amino acid；258th
Amino acid is to the 276th amino acids, totally 19 amino acid；415th amino acids to the 426th amino acids, totally 12 amino acid,
Adenovirus type XI totally two protein fragments, are the 136th amino acids to the 160th amino acids respectively, totally 25 amino acid；The
174 amino acids to the 186th amino acids, totally 13 amino acid, 14 type of adenovirus totally four protein fragments, are the 136th respectively
Amino acids are to the 160th amino acids, totally 25 amino acid；174th amino acids to the 196th amino acids, totally 23 amino
Acid；268th amino acids to the 286th amino acids, totally 19 amino acid；415th amino acids to the 450th amino acids, altogether
36 amino acid, 55 type of adenovirus totally two protein fragments, are the 136th amino acids to the 158th amino acids respectively, totally 23
Amino acid；172nd amino acids to the 184th amino acids, totally 13 amino acid, with two glycine and one between protein fragments
A serine connection, is connected into epitope chimeric protein more than one, the codon of prokaryotes preference is selected, by chimeric protein
Corresponding nucleotide sequence is translated into, is increased at the end 5' of chimeric protein gene orderNdeI restriction enzyme site increases at the end 3'
Terminator codon andEcoRI restriction enzyme site, the full-length gene order of the chemical synthesis chimeric protein；
The adenovirus type III epitope amino acid sequence of screening:
136th~151 aa:
Ile Val Thr Thr Asn Gly Asp Asn Ala Val Thr Thr Thr Thr Asn Thr
164th~187 aa:
Lys Glu Gly Leu Gln Ile Gly Lys Asp Ile Thr Thr Thr Glu Gly Glu
Glu Lys Pro Ile Tyr Ala Asp Lys
265th~284 aa:
Asp Gly Arg Asp Ala Val Ala Gly Ala Leu Ala Pro Glu Ile Val Leu
Tyr Thr Glu Asn
422nd~437 aa:
Ile Lys Val Lys Thr Asp Asp Thr Asn Gly Trp Glu Lys Asp Ala Asn
The Adenovirus type 7 epitope amino acid sequence of screening:
136th~148 aa:
Ile Val Thr Thr Gly Glu Asp Asn Ala Thr Thr Tyr Thr
161st~181 aa:
Lys Glu Gly Leu Glu Ile Gly Lys Asp Ile Thr Ala Asp Asn Lys Pro
Ile Tyr Ala Asp Lys
258th~276 aa:
Asp Gly Arg Glu Ala Ala Asp Ala Phe Ser Pro Glu Ile Val Leu Tyr
Thr Glu Asn
415th~426 aa:
Ile Lys Pro Arg Asp Thr Ala Trp Glu Lys Asp Thr
The adenovirus type XI epitope amino acid sequence of screening:
136th~160 aa:
Ile Ala Glu Gly Val Lys Asn Thr Thr Gly Glu Glu His Val Thr Glu
Glu Glu Thr Asn Thr Thr Thr Tyr Thr
174th~186 aa:
Lys Glu Gly Leu Pro Val Gly Leu Glu Val Ser Asp Glu
The 14 type epitope amino acid sequence of adenovirus of screening:
136th~160 aa:
Leu Asp Lys Gly Val Glu Thr Thr Glu Glu Arg Gln Asn Glu Asp Gly
Glu Asn Asp Glu Lys Ala Thr Tyr Thr
174th~196 aa:
Lys Asp Gly Leu Pro Ile Gly Leu Glu Val Pro Ala Glu Gly Asp Pro
Lys Pro Ile Tyr Ala Asn Lys
268th~286 aa:
Asp Leu Arg Ser Gln Lys Gln Gly Leu Lys Pro Lys Ile Val Met Tyr
Ala Glu Asn
415th~450 aa:
Ile Gly Pro Arg Thr Asp Ser Tyr Lys Glu Ile Gln Leu Asn Gly Asp
Gln Ala Trp Lys Asp Val Asn Pro Asn Gly Ile Ser Glu Leu Val Lys
Gly Asn Pro Phe
The 55 type epitope amino acid sequence of adenovirus of screening:
136th~158 aa:
Ile Ala Glu Gly Val Lys Asn Gly Glu Glu Arg Val Thr Glu Glu Glu
Asn Asn Thr Thr Thr Tyr Thr
172nd~184 aa:
Lys Glu Gly Leu Pro Ile Gly Leu Lys Val Ser Asp Glu
Adenovirus type III epitope amino acid sequence:
Ile Val Thr Thr Asn Gly Asp Asn Ala Val Thr Thr Thr Thr Asn Thr
Gly Gly Ser Lys Glu Gly Leu Gln Ile Gly Lys Asp Ile Thr Thr Thr
Glu Gly Glu Glu Lys Pro Ile Tyr Ala Asp Lys Gly Gly Ser Asp Gly
Arg Asp Ala Val Ala Gly Ala Leu Ala Pro Glu Ile Val Leu Tyr Thr
Glu Asn Gly Gly Ser Ile Lys Val Lys Thr Asp Asp Thr Asn Gly Trp
Glu Lys Asp Ala Asn
Adenovirus type 7 epitope amino acid sequence:
Ile Val Thr Thr Gly Glu Asp Asn Ala Thr Thr Tyr Thr Gly Gly Ser
Lys Glu Gly Leu Glu Ile Gly Lys Asp Ile Thr Ala Asp Asn Lys Pro
Ile Tyr Ala Asp Lys Gly Gly Ser Asp Gly Arg Glu Ala Ala Asp Ala
Phe Ser Pro Glu Ile Val Leu Tyr Thr Glu Asn Gly Gly Ser Ile Lys
Pro Arg Asp Thr Ala Trp Glu Lys Asp Thr
Adenovirus type XI epitope amino acid sequence:
Ile Ala Glu Gly Val Lys Asn Thr Thr Gly Glu Glu His Val Thr Glu
Glu Glu Thr Asn Thr Thr Thr Tyr Thr Gly Gly Ser Lys Glu Gly Leu
Pro Val Gly Leu Glu Val Ser Asp Glu
14 type epitope amino acid sequence of adenovirus:
Leu Asp Lys Gly Val Glu Thr Thr Glu Glu Arg Gln Asn Glu Asp Gly
Glu Asn Asp Glu Lys Ala Thr Tyr Thr Gly Gly Ser Lys Asp Gly Leu
Pro Ile Gly Leu Glu Val Pro Ala Glu Gly Asp Pro Lys Pro Ile Tyr
Ala Asn Lys Gly Gly Ser Asp Leu Arg Ser Gln Lys Gln Gly Leu Lys
Pro Lys Ile Val Met Tyr Ala Glu Asn Gly Gly Ser Ile Gly Pro Arg
Thr Asp Ser Tyr Lys Glu Ile Gln Leu Asn Gly Asp Gln Ala Trp Lys
Asp Val Asn Pro Asn Gly Ile Ser Glu Leu Val Lys Gly Asn Pro Phe
55 type epitope amino acid sequence of adenovirus:
Ile Ala Glu Gly Val Lys Asn Gly Glu Glu Arg Val Thr Glu Glu Glu
Asn Asn Thr Thr Thr Tyr Thr Gly Gly Ser Lys Glu Gly Leu Pro Ile
Gly Leu Lys Val Ser Asp Glu
Chimeric protein amino acid sequence:
Ile Val Thr Thr Asn Gly Asp Asn Ala Val Thr Thr Thr Thr Asn Thr
Gly Gly Ser Lys Glu Gly Leu Gln Ile Gly Lys Asp Ile Thr Thr Thr
Glu Gly Glu Glu Lys Pro Ile Tyr Ala Asp Lys Gly Gly Ser Asp Gly
Arg Asp Ala Val Ala Gly Ala Leu Ala Pro Glu Ile Val Leu Tyr Thr
Glu Asn Gly Gly Ser Ile Lys Val Lys Thr Asp Asp Thr Asn Gly Trp
Glu Lys Asp Ala Asn Gly Gly Ser Ile Val Thr Thr Gly Glu Asp Asn
Ala Thr Thr Tyr Thr Gly Gly Ser Lys Glu Gly Leu Glu Ile Gly Lys
Asp Ile Thr Ala Asp Asn Lys Pro Ile Tyr Ala Asp Lys Gly Gly Ser
Asp Gly Arg Glu Ala Ala Asp Ala Phe Ser Pro Glu Ile Val Leu Tyr
Thr Glu Asn Gly Gly Ser Ile Lys Pro Arg Asp Thr Ala Trp Glu Lys
Asp Thr Gly Gly Ser Ile Ala Glu Gly Val Lys Asn Thr Thr Gly Glu
Glu His Val Thr Glu Glu Glu Thr Asn Thr Thr Thr Tyr Thr Gly Gly
Ser Lys Glu Gly Leu Pro Val Gly Leu Glu Val Ser Asp Glu Gly Gly
Ser Leu Asp Lys Gly Val Glu Thr Thr Glu Glu Arg Gln Asn Glu Asp
Gly Glu Asn Asp Glu Lys Ala Thr Tyr Thr Gly Gly Ser Lys Asp Gly
Leu Pro Ile Gly Leu Glu Val Pro Ala Glu Gly Asp Pro Lys Pro Ile
Tyr Ala Asn Lys Gly Gly Ser Asp Leu Arg Ser Gln Lys Gln Gly Leu
Lys Pro Lys Ile Val Met Tyr Ala Glu Asn Gly Gly Ser Ile Gly Pro
Arg Thr Asp Ser Tyr Lys Glu Ile Gln Leu Asn Gly Asp Gln Ala Trp
Lys Asp Val Asn Pro Asn Gly Ile Ser Glu Leu Val Lys Gly Asn Pro
Phe Gly Gly Ser Ile Ala Glu Gly Val Lys Asn Gly Glu Glu Arg Val
Thr Glu Glu Glu Asn Asn Thr Thr Thr Tyr Thr Gly Gly Ser Lys Glu
Gly Leu Pro Ile Gly Leu Lys Val Ser Asp Glu
The DNA sequence dna of chemical synthesis chimeric protein
The building of HAdV epitope chimeric protein recombinant plasmid:
Extract plasmid pET-28a(+), it is U.S. Novagen Products, usesNdeI andEcoRI double digestion returns after electrophoresis
The plasmid large fragment for receiving digestion, is dissolved in deionized water, usesNdeIWithEcoThe chemically synthesized HAdV epitope of RI double digestion is embedding
Hop protein genetic fragment is dissolved in deionized water after electrophoresis recycling, takes DNA fragmentation after above-mentioned digestion, be inserted into carrier pET28a
In (+)NdeIWithEcoBetween the site RI, expression is containing there are five types of the chimeric proteins of type Adenovirus Antigen epitope；
The screening and identification of recombinant plasmid:
By recombinant plasmid transformed e. coli bl21 (DE3), coating contains 60 μ g/mL kanamycins LB plates, sets 37 DEG C overnight；It is secondary
Day, random picking converted bacterium colony and control bacterium, and the control bacterium is plasmid pET28a transformed bacteria, extracted plasmid, useNdeI andEcoThe verifying of RI double digestion, 1.0% agarose gel electrophoresis results show to cut the target gene fragment of 1104bp；Meanwhile it will
Plasmid containing the genetic fragment carries out DNA sequence analysis, and sequence analysis confirms that recombinant plasmid contains five kinds to link together
Type Adenovirus Antigen epitope gene segment, sequence are completely correct:
ATT GTT ACG ACG AAC GGT GAT AAT GCT GTT ACG ACG ACG ACG AAT ACG GGC GGC
TCC AAA GAA GGT CTG CAA ATC GGC AAA GAC ATC ACC ACG ACC GAA GGC GAA GAA AAA
CCG ATC TAC GCA GAT AAA GGC GGT AGT GAT GGT CGC GAT GCA GTT GCT GGT GCC CTG
GCA CCG GAA ATT GTC CTG TAT ACC GAA AAC GGC GGT AGC ATC AAA GTT AAA ACC GAT
GAC ACG AAC GGC TGG GAA AAA GAT GCG AAT GGC GGT AGC ATC GTC ACG ACC GGT GAA
GAC AAC GCC ACG ACC TAT ACC GGC GGT TCT AAA GAA GGC CTG GAA ATT GGT AAA GAT
ATC ACC GCA GAC AAT AAA CCG ATT TAC GCT GAT AAA GGC GGT AGC GAC GGT CGT GAA
GCG GCC GAT GCG TTT TCT CCG GAA ATT GTG CTG TAT ACC GAA AAT GGC GGT TCA ATC
AAA CCG CGC GAT ACC GCA TGG GAA AAA GAC ACG GGC GGT TCG ATT GCT GAA GGC GTG
AAA AAC ACG ACC GGT GAA GAA CAT GTT ACC GAA GAA GAA ACG AAT ACG ACC ACG TAC
ACC GGC GGT TCT AAA GAA GGC TTA CCG GTG GGT CTG GAA GTT AGT GAT GAA GGC GGT
TCC CTG GAC AAA GGC GTT GAA ACC ACG GAA GAA CGT CAG AAC GAA GAT GGT GAA AAT
GAC GAA AAA GCC ACC TAT ACG GGC GGT AGC AAA GAT GGT CTG CCG ATT GGC CTG GAA
GTG CCG GCC GAA GGC GAT CCG AAA CCG ATT TAT GCC AAC AAA GGC GGT AGC GAT CTG
CGT TCT CAG AAA CAA GGT CTG AAA CCG AAA ATT GTT ATG TAT GCA GAA AAC GGC GGT
TCA ATC GGC CCG CGC ACC GAT TCG TAC AAA GAA ATT CAG CTG AAT GGT GAT CAA GCT
TGG AAA GAC GTC AAC CCG AAT GGC ATC AGT GAA CTG GTG AAA GGT AAC CCG TTC GGC
GGT TCC ATT GCC GAA GGC GTC AAA AAT GGT GAA GAA CGC GTG ACC GAA GAA GAA AAC
AAT ACG ACG ACG TAC ACG GGC GGT AGC AAA GAA GGT CTG CCG ATT GGT CTG AAA GTC
TCC GAT GAA TAA
The chimeric protein of the expression of recombinant plasmid of building type Adenovirus Antigen epitope containing there are five types of, 363 amino acid of overall length,
Its N-terminal is adenovirus type III epitope protein segment, is sequentially connected in series 7 types, 11 types, 14 type epitope protein segments, C-terminal is
55 type epitope protein segment of adenovirus, each fragment length are followed successively by long 85 amino acid of 3 types, long 74 amino acid of 7 types, and 11
Type grows 41 amino acid, long 112 amino acid of 14 types, long 39 amino acid of 55 types, between protein fragments by two glycine and
One serine connection, amino acid sequence are as follows:
Ile Val Thr Thr Asn Gly Asp Asn Ala Val Thr Thr Thr Thr Asn Thr
Gly Gly Ser Lys Glu Gly Leu Gln Ile Gly Lys Asp Ile Thr Thr Thr
Glu Gly Glu Glu Lys Pro Ile Tyr Ala Asp Lys Gly Gly Ser Asp Gly
Arg Asp Ala Val Ala Gly Ala Leu Ala Pro Glu Ile Val Leu Tyr Thr
Glu Asn Gly Gly Ser Ile Lys Val Lys Thr Asp Asp Thr Asn Gly Trp
Glu Lys Asp Ala Asn Gly Gly Ser Ile Val Thr Thr Gly Glu Asp Asn
Ala Thr Thr Tyr Thr Gly Gly Ser Lys Glu Gly Leu Glu Ile Gly Lys
Asp Ile Thr Ala Asp Asn Lys Pro Ile Tyr Ala Asp Lys Gly Gly Ser
Asp Gly Arg Glu Ala Ala Asp Ala Phe Ser Pro Glu Ile Val Leu Tyr
Thr Glu Asn Gly Gly Ser Ile Lys Pro Arg Asp Thr Ala Trp Glu Lys
Asp Thr Gly Gly Ser Ile Ala Glu Gly Val Lys Asn Thr Thr Gly Glu
Glu His Val Thr Glu Glu Glu Thr Asn Thr Thr Thr Tyr Thr Gly Gly
Ser Lys Glu Gly Leu Pro Val Gly Leu Glu Val Ser Asp Glu Gly Gly
Ser Leu Asp Lys Gly Val Glu Thr Thr Glu Glu Arg Gln Asn Glu Asp
Gly Glu Asn Asp Glu Lys Ala Thr Tyr Thr Gly Gly Ser Lys Asp Gly
Leu Pro Ile Gly Leu Glu Val Pro Ala Glu Gly Asp Pro Lys Pro Ile
Tyr Ala Asn Lys Gly Gly Ser Asp Leu Arg Ser Gln Lys Gln Gly Leu
Lys Pro Lys Ile Val Met Tyr Ala Glu Asn Gly Gly Ser Ile Gly Pro
Arg Thr Asp Ser Tyr Lys Glu Ile Gln Leu Asn Gly Asp Gln Ala Trp
Lys Asp Val Asn Pro Asn Gly Ile Ser Glu Leu Val Lys Gly Asn Pro
Phe Gly Gly Ser Ile Ala Glu Gly Val Lys Asn Gly Glu Glu Arg Val
Thr Glu Glu Glu Asn Asn Thr Thr Thr Tyr Thr Gly Gly Ser Lys Glu
Gly Leu Pro Ile Gly Leu Lys Val Ser Asp Glu
Express the screening and identification of chimeric protein engineering bacteria:
The positive transformant containing recombinant plasmid is seeded in the test tube of the culture medium of LB containing 3mL, the culture medium, which contains, blocks that
Toxin 60 μ g/mL, 37 DEG C of shaken cultivation 3h add IPTG to final concentration 0.5mmol/L, continue shaken cultivation and induce 6h, centrifugation is received
Collect thallus and carry out SDS-PAGE detection, it is 55 kD's that recon, which expresses relative molecular weight,
HAdV chimeric protein, and bacterium BL21 (DE3) is compareed without this protein band；
The purifying of adenovirus hominis epitope chimeric protein:
1) the ultrasound cracking of Adenovirus Antigen epitope chimeric protein engineering bacteria is expressed
Bacterium is received into the engineering bacteria centrifugation of inducing expression chimeric protein, wherein centrifugal condition is 8000 rpm, 10 min, 4 DEG C；Thallus
Be resuspended in the bacterial lysate of 1/10 volume of original fluid, the bacterial lysate containing 20 mmol/L PB pH7.4 and
5% glycerol, ice-bath ultrasonic break bacterium, supernatant are collected by centrifugation；
2) the amine sulfate fractional precipitation of Adenovirus Antigen epitope chimeric protein is expressed
Supernatant volume accurately is measured, appropriate saturation sulfuric acid amine aqueous solution is added into supernatant, it is stirring while adding, keep the end of amine sulfate dense
Degree is 25%, is set in ice bath overnight；
Supernatant is collected by centrifugation, appropriate solid sulfur acid amide powder is added, it is stirring while adding, make final concentration of the 50% of amine sulfate, sets
In ice bath overnight；Precipitating is collected by centrifugation in next day；It being suspended and is precipitated with 200mL equilibrium liquid, the equilibrium liquid is 50mM Tris-HCl,
PH 7.5 is packed into bag filter, to 1000mL equilibrium liquid dialysed overnight, changes liquid 4 times；Supernatant is collected by centrifugation, directly upper DEAE-
The purifying of Separose FF anion column；
3) DEAE-Separose FF anion column purifies
Balance DEAE-Separose FF anion column is rinsed with equilibrium liquid, the equilibrium liquid is 50mM Tris-HCl, pH
7.5, the direct upper prop of the supernatant that then upper step has been dialysed, flow velocity is 100 mL/h；Column is sufficiently washed with equilibrium liquid after end of the sample,
Albumen successively is eluted with the solution containing 100,200,300,400,500,1000 mmol/L NaCl again, collects each eluting peak egg
It is white, with each peak albumen of 12% SDS-PAGE electrophoresis detection, determine that Adenovirus Antigen epitope of which eluting peak containing expression is chimeric
Albumen；It is Adenovirus Antigen epitope chimeric protein that 300 mmol/L NaCl, which elute protein peak,.
4. adenovirus hominis epitope chimeric protein described in claim 1 is preparing adenovirus vaccine, antibody or antigen detection
Application in reagent.
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| CN111044728B (en) * | 2019-09-27 | 2023-05-30 | 广州医科大学附属第一医院 | IgM antibody colloidal gold test strip for rapidly detecting adenovirus and preparation method thereof |
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| CN105348391A (en) * | 2015-11-03 | 2016-02-24 | 李越希 | Enteric cytopathogenic human orphan virus 6-type VP1 protein specific epitope, fusion protein of enteric cytopathogenic human orphan virus 6-type VP1 protein specific epitope and preparation method and use of fusion protein |
| CN105671212A (en) * | 2016-04-14 | 2016-06-15 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Preparation and application of adenovirus parting gene chip |
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