CN107365780A - A kind of clone of eastern subterranean termite salivary gland β glycosidase genes and prokaryotic expression method - Google Patents
A kind of clone of eastern subterranean termite salivary gland β glycosidase genes and prokaryotic expression method Download PDFInfo
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- CN107365780A CN107365780A CN201710680489.5A CN201710680489A CN107365780A CN 107365780 A CN107365780 A CN 107365780A CN 201710680489 A CN201710680489 A CN 201710680489A CN 107365780 A CN107365780 A CN 107365780A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2445—Beta-glucosidase (3.2.1.21)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01021—Beta-glucosidase (3.2.1.21)
Abstract
The invention discloses a kind of clone of eastern subterranean termite salivary gland β glycosidase genes and prokaryotic expression method.The present invention designs degenerate pcr sequence by other termite kind salivary gland β glycosidases protein sequences provided on NCBI, amplification obtains the Partial Fragment of target gene, then the full length cDNA sequence of eastern subterranean termite salivary gland β glycosidase genes is obtained by 3 ' and 5 ' RACE methods；The a length of 1691bp of cDNA sequence, ORFs size are 1488bp, and protein sequence has typical glycosyl hydrolase I families conserved domain, have 95% uniformity with North America reticulitermes flavipe salivary gland β glucosides enzyme amino acid sequence.On this basis, expand the gene open reading frame sequence, construction expression plasmid pET32a GZRfbg1, after be transferred in Escherichia coli Rosetta 2 (DE3) and BL21, induced expression is carried out using IPTG, finally successful fusion gives expression to β glucosides zymoproteins in Rosetta 2 (DE3) cell.The inventive method can be used for the structure of the development of resources of termite enzyme and following termite Bionic digestion system, have good application value.
Description
Technical field
The present invention relates to biological technical field, and in particular to a kind of clone of eastern subterranean termite salivary gland beta -glycosidase gene
And prokaryotic expression method.
Background technology
Lignocellulosic is renewable resource the abundantest in terrestrial ecosystem, and its biological conversion is that new energy is opened
The frontier of hair.Eastern subterranean termite (food wood) is one of most important termite population of south China, its internal lignocellulosic
Enzyme system is up to more than 97% to the degradation efficiency of lignocellulosic, and the system is considered as that 21 century most promising biology is anti-
Answer device.
Termite cellulase can mainly be included by the microorganism secretion of itself salivary gland, middle intestines and hindgut：Inscribe Portugal gathers
Carbohydrase, beta -glycosidase and exoglucanase.Up to the present, because termite does not have complete whole genome sequence, therefore
Acquisition for termite its own cellulose enzyme gene, rely primarily on library screening, protein purification backward sequencing and degenerate pcr
With reference to cDNA RLM-RACE technologies.Wherein degenerate pcr combination cDNA end technologies are the quickest, convenient acquisition full-length cDNA sequences
The method of row.The expression of termite its own cellulose gene possesses some special knowledge in Escherichia coli, fungi, insect expression system,
Wherein escherichia coli prokaryotic expression system is the research meanses of protein expression research high-efficiency and economic the most.
The content of the invention
It is an object of the invention to：Clone and the protokaryon of a kind of eastern subterranean termite salivary gland beta -glycosidase gene are provided first
Expression.
In order to realize above-mentioned purpose of the present invention, the invention provides a kind of eastern subterranean termite salivary gland beta -glycosidase gene
(GZRfbg1), its cDNA sequence is made up of 1691 base-pairs, and ORFs size is 1488bp, translation initiation codon
ATG is located at the 45th~47 base, and terminator codon TAA is located at the 1530th~1532, has poly- at 3 ' ends
Adenylation signal sequences AATAAA and poly (A+) tail feature sequence, such as SEQ ID NO:Shown in 1.
Present invention also offers a kind of eastern subterranean termite salivary gland beta -glycosidase GZRfBG1 albumen, it is by described yellow chest
The ORFs of reticulitermes flavipe salivary gland beta -glycosidase gene (GZRfbg1) encodes 448 amino acid, and albumen size is
56.45kDa, signal peptide shearing site between the 17th and the 18th amino acid there is typical glycosyl hydrolase I families to protect
Keep domain.
The acquisition methods of above-mentioned eastern subterranean termite salivary gland beta -glycosidase gene (GZRfbg1) cDNA sequence of the present invention include
Following steps：Total serum IgE is extracted with eastern subterranean termite, other termite kind salivary gland beta -glycosidase protein provided on NCBI are provided
Sequences Design degenerate pcr sequence, amplification obtain the part piece of the eastern subterranean termite salivary gland beta -glycosidase gene (GZRfbg1)
Section, then obtain full length cDNA sequence by 3 ' and 5 ' RACE methods；Wherein, the degenerate pcr sequence such as SEQ ID NO:2~11 institutes
Show；The primer used in the RACE methods such as SEQ ID NO:Shown in 12~22.
Present invention also offers a kind of preparation method of the restructuring GZRfBG1 albumen based on escherichia expression system, its
Comprise the following steps：
(1) gene open reading frame sequence expands：According to the eastern subterranean termite salivary gland beta -glycosidase gene
(GZRfbg1) cDNA sequence, design primer removes signal peptide sequence and terminator codon, and draws respectively in upstream and downstream
NcoI and XhoI Restriction Enzyme enzyme cutting restriction enzyme sites are introduced in thing；
(2) amplified fragments are connected into pMD 20-T plasmids using pMD 20-T carriers connection kit, converts Escherichia coli
DH5 α competent cells, obtain restructuring amplification bacterial strain pMD-20T-GZRfbg1-DH5 α；
(3) respectively by restructuring amplification bacterial strain pMD-20T-GZRfbg1-DH5 α and expression plasmid pET32a empty carriers NcoI
With the fast enzyme cutting double digestions of XhoI, digestion products are attached with T4 ligases afterwards after purification, convert bacillus coli DH 5 alpha competence
Cell, obtain recombinant expression plasmid amplification bacterial strain pET32a-GZRfbg1-DH5 α；
(4) recombinant expression plasmid is expanded into bacterial strain pET32a-GZRfbg1-DH5 α conversion E. coli expression strains
Rosetta 2 (DE3), obtain GZRfBG1 expression bacterial strain pET32a-GZRfbg1-Rosetta 2 (DE3)；
(5) bacterial strain pET32a-GZRfbg1-Rosetta 2 (DE3), 1% inoculation LB culture mediums, in 25 DEG C of conditions will be expressed
Under, 220r/min shaking table cultures to OD600For 0.4~0.5, using final concentration of 0.1mmol/L IPTG, continue in 20 DEG C of inductions
After culture 18 hours, 5min is centrifuged under the conditions of 4 DEG C, 8000 × g, collects cell precipitation；
(6) cell precipitation is suspended again with 50mmol/L Tris-HCl buffer solutions (pH 8.0), added final concentration of
5mmol/L EDTA and 1mmol/l PMSF, piping and druming is uniform, then ultrasonic treatment is carried out under condition of ice bath, amplitude 50%, work
Stop 5s as 3s, continue 20min；Then cell is centrifuged into 20min under the conditions of 4 DEG C, 8000 × g, obtains supernatant as solubility
GZRfBG1 albumen, it is precipitated as the GZRfBG1 albumen of inclusion body state.
Wherein, the upstream primer sequence such as SEQ ID NO:Shown in 23, restriction enzyme site CCATGG；The anti-sense primer
Sequence such as SEQ ID NO:Shown in 24, restriction enzyme site CTCGAG.
Relative to prior art, the invention has the advantages that and beneficial effect：
Salivary gland β-glucosides that the present invention is cloned from eastern subterranean termite for the first time using degenerate pcr combination RACE method
The cDNA full length sequences of enzyme gene, by being connected structure recombinant expression plasmid with pET32a, select Escherichia coli Rosetta 2
(DE3) as recombinant protein production cell, the codon preference that eukaryotic source albumen is expressed in prokaryotic expression system is overcome
Property.The present invention supplements important theoretical foundation for the development of resources of termite enzyme and following termite Bionic digestion system constructing, has
There is good application value.
Brief description of the drawings
Fig. 1 is eastern subterranean termite total serum IgE gel electrophoresis qualification figure, wherein, swimming lane M is DNA Marker (DL2000), 1 He
2 swimming lanes are Total RNAs extraction thing.
Fig. 2 is eastern subterranean termite salivary gland beta -glycosidase gene GZRfbg1 degenerate pcr figure, and wherein swimming lane M is DNA
Marker (DL2000), 1~5 swimming lane are GZRfbg1 gene fragment amplification products after the combination of different degenerate primers.
Fig. 3 is recombinant plasmid pET32a-GZRfbg1 digestion qualification result figures, and wherein M is DNA Marker (Marker
4)；Swimming lane 1,3 is recombinant expression carrier pET32a-GZRfbg1 NcoI and XhoI double digestion positive identification results.
Fig. 4 is homologous comparison result of the protein amino acid sequence of GZRfbg1ORF codings on NCBI.Conserved amino
Acid is divided into three level identities, wherein black, and dark-grey and grey represents 100%, 80% and 60% conservative respectively.
Fig. 5 is the SDS-PAGE and western- of eastern subterranean termite salivary gland beta -glycosidase amalgamation and expression in Escherichia coli
Blot qualification figures, wherein, A is SDS-PAGE results, and wherein swimming lane M is albumen (invitrogen companies), and swimming lane 1 is empty carrier
The cell holoprotein sample of pET32a expression in Escherichia coli Rosetta 2 (DE3), swimming lane 2 are GZRfBG1 in Escherichia coli
The cell holoprotein sample expressed in BL21, swimming lane 3 are the cell of GZRfBG1 expression in Escherichia coli Rosetta 2 (DE3)
Holoprotein sample, swimming lane 4 are that the cell of GZRfBG1 expression in Escherichia coli Rosetta 2 (DE3) cracks supernatant protein sample
Product, swimming lane 5 are GZRfBG1 amalgamation and expression albumen after purification；B is western-blot results, and wherein swimming lane M is albumen
(invitrogen companies), swimming lane 1 are the protein sample of pET32a empty carriers expression in Escherichia coli Rosetta 2 (DE3)；
Swimming lane 2 is protein precipitation sample after the cell of GZRfBG1 expression in Escherichia coli Rosetta 2 (DE3) cracks；Swimming lane 3 is
The cell of GZRfBG1 expression in Escherichia coli Rosetta 2 (DE3) cracks supernatant protein sample, and swimming lane 4 is after purification
GZRfBG1 amalgamation and expression albumen.
Embodiment
In order that the purpose of the present invention, technical scheme and advantageous effects become apparent from, with reference to embodiments, to this
Invention is further elaborated.It should be appreciated that the embodiment described in this specification is just for the sake of this hair of explanation
Bright, being not intended to limit the present invention, the parameter of embodiment, ratio etc. can suit measures to local conditions to make a choice and have no substance to result
Influence.
Embodiment 1
1) extraction of total serum IgE and total cDNA amplification
After the eastern subterranean termite of Long-term breeding in laboratory is rinsed with 0.01mM phosphate buffer (PB, pH 7.0),
Using the E.Z.N.A. of OMEGA companiesTMTotal RNA Kit II total RNA extraction reagents boxes extract eastern subterranean termite total serum IgE
(Fig. 1).It is total using the PrimeScriptTMII 1st Strand cDNA Synthesis Kit reverse transcriptions of Takara companies
RNA obtains the total cDNA of eastern subterranean termite.
2) degenerate pcr obtains beta -glycosidase genetic fragment
Use online software iCODEHOP (http://blocks.fhcrc.org/codehop.html), according to termite saliva
Liquid gland beta -glycosidase homologous protein sequence designs degenerate pcr amplimer.Carried out using the Premix Ex Taq of TAKARA companies
Degenerate pcr expands (Fig. 2).
Degenerate pcr amplimer is as shown in table 1.
The degenerate pcr amplimer sequence of table 1
R=A/G, Y=C/T, M=A/C, K=G/T, S=C/G, W=A/T, H=A/C/T, B=C/G/T, V=A/C/G,
D=A/G/T, N=A/C/G/T.
Degenerate pcr reaction system is prepared according to table 2.
The degenerate pcr reaction system of table 2
After mixing.Reaction condition：First stage：94 DEG C of 1min, 1 circulation；Second stage：94 DEG C of 30s, 50 DEG C of 30s, 72
DEG C 30s, totally 30 circulations；Phase III：72 DEG C of extension 10min；Last 4 DEG C of preservations.
PCR amplifications finish, and take 5 μ L products to be detected on 1.0% Ago-Gel, select big DNA cloning fragment to send
Genome company is sequenced.
3) RACE amplifications obtain beta -glycosidase full length gene sequence
After obtaining beta -glycosidase gene fragment order by degenerate pcr, Invitrogen companies are used on this basis
3 ' RACE System for Rapid Amplification of cDNA Ends kits and the 5 ' of GIBCOBRL companies
RACE System for Rapid Amplification of cDNA Ends kits carry out 3 ' RACE and 5 ' RACE respectively
Amplified reaction, it is therefore intended that obtain eastern subterranean termite salivary gland beta -glycosidase full length gene cDNA sequence.Universal
Amplification primer (UAP), Abridged Universal Amplification primer (AUAP)
(Invitrogen supplied) and GSP primers are used to expand cDNA3'- ends.Abridged Anchor primer(AAP)
(Invitrogen supplied) and GSP primers are used to expand cDNA5'- ends.Finally with overlapping extension by cDNA5'- and
3'- ends connect.
The primer used of RACE amplifications is as shown in table 3.
The primer used of table 3RACE amplifications
Expanded by 13 ' Race- of wheel and 35 ' Race of wheel PCR, after each step amplified fragments send company to be sequenced, according to
Sequences Design gene specific primer, finally expand and obtain the eastern subterranean termite beta -glycosidase gene cDNA sequence of total length, and
Online BLAST is carried out on NCBI and compares identification, the salivary gland beta -glycosidase unnamed gene that the eastern subterranean termite clones is
GZRfbg1, its full length cDNA sequence a length of 1691bp, ORFs ORF size is 1488bp, translation initiation codon ATG
Positioned at the 45th~47 base of cDNA, terminator codon TAA is located at the 1530th~1532, has poly- in 3 '-terminal
Adenylation signal sequences AATAAA and poly (A+) tail feature sequence；GZRfbg1 full length cDNA sequences such as SEQ ID
NO:Shown in 1.
GZRfbg1 ORF encodes 448 amino acid, and prediction albumen size is 56.45kD, and signal peptide shearing site is located at
Between 17th and 18 amino acid, there is typical glycosyl hydrolase I families conserved domain.GZRfBG1 is eukaryotic
The beta -glycosidase in source, the protease amino acid sequence with from North America reticulitermes flavipe, Workers of Coptotermes formosanus Shiraki, yellow-wing subterranean termite and
Nasutitermes takasagoensis salivary gland beta -glycosidase sequence, respectively with 95%, 86%, 83%, 81%
Uniformity, its BLAST comparison result on NCBI are shown in Fig. 4.
4) gene open reading frame (ORF) sequence amplification
According to beta -glycosidase gene GZRfbg1cDNA sequences, using DNASTAR software Design primers, signal peptide sequence is removed
Row and terminator codon, and NcoI and XhoI Restriction Enzyme enzyme cutting restriction enzyme sites are introduced in upstream and anti-sense primer respectively：
Sense primer and anti-sense primer are as follows：
Upstream primer sequence such as SEQ ID NO:Shown in 23；
Downstream primer sequence such as SEQ ID NO:Shown in 24.
Using, total cDNA, Premix Ex Taq PCR enzymatic amplification beta -glycosidase genes ORF.With 1.0% Ago-Gel
Reclaimed after electrophoretic separation with Omega glue reclaims kit.It will be reclaimed using Takara companies pMD 20-T carriers connection kit
Fragment connects pMD 20-T plasmids, structure amplification plasmid pMD-20T-GZRfbg1；Reference《Molecular cloning III》Methods described is entered
Prepared by row bacillus coli DH 5 alpha, competence, ampicillin plate screening is carried out after conversion plasmid pMD-20T-GZRfbg1；
Bacterium colony PCR identification positive clone molecules are carried out with sterilizing toothpick picking single bacterium colony in super-clean bench, obtain gene fragment amplification bacterial strain
pMD-20T-GZRfbg1-DH5α；Positive colony single bacterium colony is seeded in liquid LB and carries out plasmid amplification.With reference to Omega companies
The extraction and purification kit specification extraction plasmid of Plasmid Mini Kit I DNAs.By multiple positive clone molecule matter
Li Song genome companies are sequenced, and the uniformity of extension increasing sequence and cDNA sequence are compared using DNASTAR softwares, it is ensured that ORF codons
To frame correctly and without amplification mutation.
5) pET32a-GZRfbg1 recombinant plasmids are built
Respectively will amplification plasmid pMD-20T-GZRfbg1 and expression vector pET32a empty carriers NcoI and the fast enzyme cuttings of XhoI
Double digestion, the carrier after gel electrophoresis and target gene fragment is separately recovered using OMEGA companies glue reclaim kit, after use T4
Ligase, normal temperature connection more than 30min, converts DH5 α competent cells, carries out ampicillin plate screening, picking monoclonal
Bacterium, which is put into LB culture mediums, shakes bacterium, and bacterium solution PCR identifies positive colony, obtains gene fragment amplification bacterial strain pET-32a-
GZRfbg1-DH5α.Expand culture, extraction recombinant plasmid pET32a-GZRfbg1 (Fig. 3) after sequencing is correct；
6) GZRfBG1 recombinant proteins prokaryotic expression and identification
By expression plasmid pET32a-GZRfbg1 and pET32a empty carriers convert E. coli expression strains BL21 with
Rosetta 2 (DE3), obtain GZRfBG1 BL21 and Rosetta 2 (DE3) expression bacterial strain.Two kinds of expression bacterial strains 1% are connect
Kind contains 100 μ g/ml ampicillin LB culture mediums, under the conditions of 37 DEG C, 220r/min shaking table cultures to OD600For 0.4, use
Final concentration of 0.5mmol/L IPTG inducible proteins expression, after continuing culture 3 hours, is centrifuged under the conditions of 4 DEG C, 8 000 × g
5min, collect cell precipitation.
A) part cell precipitation is suspended again with 50mmol/L Tris-HCl buffer solutions (pH 8.0), adds final concentration
It is uniform for 5mmol/L EDTA and 1mmol/l PMSF, piping and druming.Cell solution is subjected to ultrasonic treatment under condition of ice bath
Cell after (amplitude 50%, work 3s stop 5s, continue 20min) ultrasonic degradations centrifuges under the conditions of 4 DEG C, 8 000 × g
20min, supernatant (soluble recombinant protein) and precipitation (non-solubility recombinant protein) are added into 2 × SDS-PAGE loading buffers
Liquid, sample 5min is boiled in boiling water.
B) another part cell precipitation is directly suspended again with 2 × SDS-PAGE sample-loading buffers, sample is boiled in boiling water
5min, detect total protein concentration.Reference《Molecular cloning III》Method carries out SDS-PAGE and Western-blot identifications GZRfbg1
Expression (Fig. 4).Western-blot uses PVDF electricity transferring films, and primary antibody uses the anti-His-tag of mouse (MBL, Japan), secondary antibody
Anti- mouse IgG (MBL, Japan) immune detections selection DAB Western Blotting detection kits are connected using HRP-
(CWBIO, China) supernatants and the restructuring for obtaining merging Trx.tag, S.tag and 6 × His-tag protein tags in precipitation
Eastern subterranean termite salivary gland beta -glycosidase GZRfBG1, size are about 74kDa, and label protein size is about 20kDa, and Trx.tag is helped
Molten albumen, 6 × His tag are following protein purification label (Fig. 5).
The announcement and teaching of book according to the above description, those skilled in the art in the invention can also be to above-mentioned embodiment party
Formula carries out appropriate change and modification.Therefore, the invention is not limited in embodiment disclosed and described above, to this
Some modifications and changes of invention should also be as falling into the scope of the claims of the present invention.In addition, although this specification
In used some specific terms, but these terms are merely for convenience of description, do not form any restrictions to the present invention.
Sequence table
SEQ ID NO:1 GZRfbg1 full length cDNA sequences
TATTCTGACATTCTCACGTCTAGGGCGCTTGACAGAGCAACAAGATGAGGTTACAGACGGTTTGCTTCGTCAT
CTTTGTGACGGCAGTATTCGGGGCTGACGTCGATAACGAAACCCTCGTCACGTTTCCTAAAGACTTTAAGTTAGGCG
CCGCTACGGCTTCATACCAGATTGAAGGAGGATGGAATGAGGATGGAAAGGGTGTCAATATTTGGGACACACTGACG
CATGAGCGCTCACAATTAGTGGTTGATAAATCAAGCGGTGACGTGGCTGACGACTCGTATCATCTTTATATGGAGGA
TGTGCAGCTTCTGAAGAACATGGGGGCACAAATTTATCGCTTCTCTATATCTTGGGCTCGCATCCTGCCTGAAGGAC
ATGATAATAAGGTGAACCAGGCGGGCATTGATTACTACAACAAGCTCATAGACGCACTTCTAGACAATGAAATAGAG
CCGATGGTTACTATGTATCACTGGGATCTACCCCAGACACTCCAAGACCTGGGAGGATGGCCAAATAGAGAATTGGC
AAAATACTCCGAGAATTACGCCCGCGTTTTATTTAAAAACTTTGGAGACCGGGTTAAATTGTGGCTCACATTCAACG
AGCCTCTGACTTTCATGGATGCATATGCATCTGAGACAGGAATGGCTCCATCAATTGACACACCCGGTATCGGCGAC
TACCTTGCGGCACACACTGTGATCCTTGCCCATGCCAATATCTACCGTATGTATGAGAGGGAATTCAAAGCGGAtCA
GcAAGGAGAGGTTGGTATCGCACTCAACATACACTGGTGTGAGCCGGTGACTAATTCTACAGAGGACgTTGAGGCTT
GTGAAAGGTATCAACAGTTCAATCTGGGAATATACGCTCATCCCATCTTCTCTGAAGAGGGCGATTACCCCAGTGTT
TTGAAAGCGAGGGTAGACGCAAACAGCGCAGCGGAAAATTACACCACATCCCGTCTACCAAAATTCACTCCAGAtGA
AGTAGATTTCATCAGAGGAACACATGATTTCTTGGGTCTGAATTTCTACACTGCTGTAACGGGAGCTGGTGGAGTTG
AAGGGGAACCCCCGTCGCGGTACAGAGACGCGGGCGCGATCATATCACAGGATCCGGACTGGCCCGAGTCTGCTTCT
TCATGGCTCAGAGTTGTACCATGGGGATTCCGCAAGGAACTCAACTGGATCGCGAACGAATACGGTAACCCTCCTAT
AtTCATCACTGAAAACGGCTTCTCCGACTACGGTGGCCTCAATGATACAGACAGAGTGCTGTACTACACTGAACATT
TAAAGGAGATGCTGAAGGCAATTCACATAGATGAAGTTAACGTAGTCGGATACACAGCCTGGAGCCTAGTAGACAAT
TTCGAATGGCTGCGAGGATATACTGAGAGGTTCGGTATACATGAAGTGAATTTCAACGACCCAAGTCGCCCACGAGT
TCCCAAGGAGTCAGCCAAGGTGCTCACAGAGATCTTCAAAACAAGGAGGATTCCAGAACGCTTCCTAGACTAACTTC
ATATTCGAAACGCAGAGACTTATATCAAAAATTAATTTAAAAGAGGGCTTACTGCTGACTGTGAGTTCCCTCAAAAC
AGCAATAAGGTTTATGATCATGGAAAACACTTCGAATTAATAAACTTATATACAAATATAAAAAAAAAAAAAAAAAA
A
SEQ ID NO:2 degenerate pcr amplimer F1
GGAAAGGAGTTAATATHTGGGAYAC
SEQ ID NO:3 degenerate pcr amplimer F2
TACCCCGGGGATCGGNGAYTAYYT
SEQ ID NO:4 degenerate pcr amplimer F3
ACTTCTTTGGCATGAAYTWYTAYAC
SEQ ID NO:5 degenerate pcr amplimer R1
TANATRGTRACCCTGGACGGCGT
SEQ ID NO:6 degenerate pcr amplimer R2
ATRGTRACCCTGGACGGCGTCTTTG
SEQ ID NO:7 degenerate pcr amplimer R3
CCNYTRATRGGAAGGCATCACTTTC
SEQ ID NO:8 degenerate pcr amplimer R4
CTYCCNAWRTGATGGAGAGCCGAT
SEQ ID NO:9 degenerate pcr amplimer R5
TTRACCTADYKTTTGCTCATACCGT
SEQ ID NO:10 degenerate pcr amplimer R6
ATRATRTGNCTCGTGTACTTCCTCT
SEQ ID NO:11 degenerate pcr amplimer R7
CTRTTRAARCTCACCGACGCTCCC
SEQ ID NO:12 R.B1B7-3′Race-sp1
CGGAATTCCAGTGTTTTGAAAGCGAGGGTAGAC
SEQ ID NO:13 R.B1B7-3′Race-sp2
CGGAATTCGCCTGGATTGAGCCGGTGACTAA
SEQ ID NO:14 AP （Kit is general）
GGCCACGCGTCGACTAGTACT TTTTTTTTTTTTTTTT
SEQ ID NO:15 AUAP（Kit is general）
GGCCACGCGTCGACTAGTAC
SEQ ID NO:16 R.B1B7-5′Race-sp1
CGGAATCCCCATGGTACAACTCTG
SEQ ID NO:17 R.B1B7-5′Race-sp2
TGAATTTTGGTAGACGGGATGTGG
SEQ ID NO:18 5′Race abridged a（Kit is general）
GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG
SEQ ID NO:19 5′Race abridged anchor（Kit is general）
GGCCACGCGTCGACTAGTAC
SEQ ID NO:20 R.B1B7-5′Race-sp4
CTCGCTTTCAAAACACTGGGGTAA
SEQ ID NO:21 R.B1B7-5′Race-sp5
AACCTCTCCTTTCTGATCCGCTTTGA
SEQ ID NO:22 R.B1B7-5′Race-sp6
GTAACCATCGGCTCTATTTCATTGTC
SEQ ID NO:23 sense primers
CCATGGGACGTCGATAACGAAACCCT
SEQ ID NO:24 anti-sense primers
CTCGAGGTCTAGGAAGCGTTCTGGAAT
Claims (5)
1. a kind of eastern subterranean termite salivary gland beta -glycosidase gene (GZRfbg1), it is characterised in that its cDNA sequence is by 1691
Base-pair forms, and ORFs size is 1488bp, and translation initiation codon ATG is located at the 45th~47 base, terminates close
Numeral TAA is located at the 1530th~1532, has poly-adenylation signal sequence AATAAA and poly (A+) at 3 ' ends
Tail feature sequence, such as SEQ ID NO:Shown in 1.
2. a kind of eastern subterranean termite salivary gland beta -glycosidase GZRfBG1 albumen, it is characterised in that as the Huang described in claim 1
The ORFs of chest reticulitermes flavipe salivary gland beta -glycosidase gene (GZRfbg1) encodes 448 amino acid, and albumen size is
56.45kDa, signal peptide shearing site between the 17th and the 18th amino acid there is typical glycosyl hydrolase I families to protect
Keep domain.
3. the acquisition methods of eastern subterranean termite salivary gland beta -glycosidase gene (GZRfbg1) cDNA sequence described in claim 1,
It is characterised in that it includes following steps：Total serum IgE is extracted with eastern subterranean termite, other termite kind salivas provided on NCBI are provided
Gland beta -glycosidase protein sequence designs degenerate pcr sequence, and amplification obtains the eastern subterranean termite salivary gland beta -glycosidase gene
(GZRfbg1) Partial Fragment, then obtain full length cDNA sequence by 3 ' and 5 ' RACE methods；Wherein, the degenerate pcr sequence is such as
SEQ ID NO:Shown in 2~11；The primer used in the RACE methods such as SEQ ID NO:Shown in 12~22.
4. a kind of preparation method of the restructuring GZRfBG1 albumen based on escherichia expression system, it is characterised in that including as follows
Step：
(1) gene open reading frame sequence expands：According to the eastern subterranean termite salivary gland beta -glycosidase gene (GZRfbg1)
CDNA sequence, design primer removes signal peptide sequence and terminator codon, and is introduced respectively in upstream and anti-sense primer
NcoI and XhoI Restriction Enzyme enzyme cutting restriction enzyme sites；
(2) amplified fragments are connected into pMD 20-T plasmids using pMD 20-T carriers connection kit, converts bacillus coli DH 5 alpha
Competent cell, obtain restructuring amplification bacterial strain pMD-20T-GZRfbg1-DH5 α；
(3) respectively will restructuring amplification bacterial strain pMD-20T-GZRfbg1-DH5 α and expression plasmid pET32a empty carriers with NcoI with
The fast enzyme cutting double digestions of XhoI, digestion products are attached with T4 ligases afterwards after purification, and conversion bacillus coli DH 5 alpha competence is thin
Born of the same parents, obtain recombinant expression plasmid amplification bacterial strain pET32a-GZRfbg1-DH5 α；
(4) recombinant expression plasmid is expanded into bacterial strain pET32a-GZRfbg1-DH5 α conversion E. coli expression strains Rosetta 2
(DE3) GZRfBG1 expression bacterial strain pET32a-GZRfbg1-Rosetta 2 (DE3), are obtained；
(5) bacterial strain pET32a-GZRfbg1-Rosetta 2 (DE3) will be expressed, 1% is inoculated with LB culture mediums, under the conditions of 25 DEG C,
220r/min shaking table cultures are to OD600For 0.4~0.5, using final concentration of 0.1mmol/L IPTG, continue in 20 DEG C of induction trainings
After supporting 18 hours, 5min is centrifuged under the conditions of 4 DEG C, 8000 × g, collects cell precipitation；
(6) cell precipitation is suspended again with 50mmol/L Tris-HCl buffer solutions (pH 8.0), adds final concentration of 5mmol/
L EDTA and 1mmol/l PMSF, piping and druming is uniform, then ultrasonic treatment is carried out under condition of ice bath, amplitude 50%, and work 3s stops
5s, continue 20min；Then cell is centrifuged into 20min under the conditions of 4 DEG C, 8000 × g, it is soluble g ZRfBG1 eggs to obtain supernatant
In vain, it is precipitated as the GZRfBG1 albumen of inclusion body state.
5. the preparation method of the restructuring GZRfBG1 albumen according to claim 4 based on escherichia expression system, it is special
Sign is, the upstream primer sequence such as SEQ ID NO:Shown in 23, restriction enzyme site CCATGG；The downstream primer sequence is such as
SEQ ID NO:Shown in 24, restriction enzyme site CTCGAG.
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