CN101054569A - Gene engineering bacterium, preparation and use thereof - Google Patents
Gene engineering bacterium, preparation and use thereof Download PDFInfo
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- CN101054569A CN101054569A CNA2006100279299A CN200610027929A CN101054569A CN 101054569 A CN101054569 A CN 101054569A CN A2006100279299 A CNA2006100279299 A CN A2006100279299A CN 200610027929 A CN200610027929 A CN 200610027929A CN 101054569 A CN101054569 A CN 101054569A
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Abstract
The present invention relates to a gene engineered bacteria, and its preparation method and uses, belongs to biological engineering field. The gene engineered bacteria is composed of plasmid and host bacteria in which host bacteria is any one of Ecoli DH5alpha,BL21(DE3) or Rosetta-gami(DE3), the recombinant plasmid is pET32a(+) containing gene GAD65. The construction method of engineered bacteria comprise: designing Nest-PCR upper-stream primer and down-stream primer, amplifying human GAD65 gene fragment using PCR from human pancreas cDNA library, transforming Ecoli via vector and obtaining highly effective expression recombinant human GAD65 gene engineered bacteria. The inventive engineered bacteria can be used to produce soluble active thioredoxin- human Glutamic Decarboxylase 65 fusion protein and active recombinant human GAD65 which has enzymatic activity and immunological activity and are easy to purify. The expression volume is high and cost is low .
Description
Technical field
The present invention relates to a kind of genetic engineering bacterium, especially a kind of genetic engineering bacterium that can efficiently express human glutamic acid decarboxylase 65 or Trx-human glutamic acid decarboxylase 65 fusion roteins, and the preparation method and its usage of this genetic engineering bacterium, belong to technical field of bioengineering.
Background technology
Diabetes are a kind of serious worldwide diseases, and sickness rate is high always, and whole world diabetic subject sum is nearly 200,000,000 at present, and estimating 2010 and will rise to 2.2 hundred million, 2025 will be above 300,000,000.In this huge diabetic population, have 10% to be the type i diabetes patient approximately, and great majority are young adult.Also some is adult invisible autoimmune diabetes patient (LADA) among all the other type ii diabetes crowds of 90%, these patients have the tendency that changes type i diabetes from type ii diabetes into, diagnose out these LADA patients will help the patient to carry out early stage interference treatment from the type ii diabetes crowd.
L-Glutamic decarboxylase 65 (Glutamate Decarboxylase 65, below with slightly being written as " GAD65 ") be a kind of enzyme that the decarboxylation of specificity catalysis L-L-glutamic acid generates γ-An Jidingsuan, form by 585 amino acid, molecular weight is about 65KD, extensively be present in maincenter and the peripheral nervous system, expression is also arranged in pancreas islet, testis, ovary, thymus gland and stomach, and in human pancreas islet, have only GAD65 to express.Discover: type i diabetes is that a kind of inheritance susceptible individuality causes beta Cell of islet destructive autoimmune disorder by the immune response that autoantigen mediates, GAD65 is the initiating target antigen of this immune response crux, at the antibody GAD65-Ab of GAD65 as far back as the type i diabetes clinical episodes several years ago or the more than ten years just occur.Up to now, GAD65-Ab is one effective and the most special in the diagnosis type i diabetes immune indexes.
So the existence that detects GAD65-Ab with GAD65 can be used as prediction and the diagnostic means of T1DM; Can be applicable to from T2DM patient, identify invisible T1DM patient, make the patient can carry out the early intervention treatment; Also can be used as a kind of generaI investigation means, be used for finding the high risk population of T1DM.
At present, it is unpractical adopting the method for from natural tissues separation and purification and artificial chemosynthesis to obtain a large amount of people GAD65 protein.There has been the investigator to attempt in expression systems such as rabbit reticulocyte, yeast cell, insect cell and intestinal bacteria, attempting expressing natural recombinant human GAD65 abroad, but find to have following shortcoming: (1) people GAD65 expression amount is very low, and GAD65-Ab detection method equipment requirements height only can be used for lab analysis; (2) expression of people GAD65 is mainly with the inclusion body formal representation, and is difficult to carry out correct renaturation, causes protein not have the enzyme activity and the special immunocompetence at type i diabetes; (3) time and effort consuming spends hugely, is not suitable for China's actual conditions.Based on above several aspects, press for development production domesticization high performance cheap can widespread use the method for preparing people GAD65.
Summary of the invention
The object of the present invention is to provide a kind of genetic engineering bacterium that can be used to prepare people GAD65 or Trx-human glutamic acid decarboxylase 65 fusion roteins.
The preparation method of the genetic engineering bacterium that another object of the present invention is to provide with low cost, being used to of efficiently expressing prepares people GAD65 or Trx-human glutamic acid decarboxylase 65 fusion roteins.
A further object of the present invention is to provide the purposes of the genetic engineering bacterium that can produce people GAD65, and the method for this genetic engineering bacterium production people GAD65 or Trx-human glutamic acid decarboxylase 65 fusion roteins is provided.
The genetic engineering bacterium that can be used to prepare people GAD65 or Trx-human glutamic acid decarboxylase 65 fusion roteins of the present invention is made of recombinant plasmid and host bacterium, wherein the host bacterium refers to any one among bacillus coli DH 5 alpha, BL21 (DE3) or the Rosetta-gami (DE3), and recombinant plasmid refers to the plasmid pET-32a (+) that contains the GAD65 gene.
Genetic engineering bacterium provided by the invention is further characterized in that: described recombinant plasmid is pET-32a (+)-P
T7-TrxA-6 * His-GAD65, wherein, P
T7It is the promotor of described plasmid pET-32a (+); TrxA is a Trx; 6 * His is the Histidine purification tag.
The construction process of the genetic engineering bacterium of a kind of efficiently expressing recombinant human GAD65 provided by the invention, comprise design Nest-PCR upstream and downstream primer, with the PCR people GAD65 gene segment that from human pancreas cDNA storehouse, increases, and be transformed into intestinal bacteria by expression vector, it is characterized in that the PCR primer that designs contains the enteropeptidase restriction enzyme site, people GAD65 gene fragment process restricted type endonuclease digestion with pcr amplification, be cloned among the expression plasmid pET-32a (+), be built into recombinant plasmid pET-32a-GAD65, among the transformed into escherichia coli BL21 (DE3), obtain the genetic engineering bacterium of efficiently expressing recombinant human GAD65.
Particularly, the construction process of genetic engineering bacterium of the present invention, the concrete operations step is as follows:
The first step primer design is with synthetic
CDNA sequence according to known person GAD65, design specific Nest-PCR upstream primer A1, A2 and downstream primer B1, wherein introduce KpnI restriction enzyme digestion sites and enteropeptidase restriction enzyme site among the upstream primer A2, introduce the BamHI restriction enzyme digestion sites among the downstream primer B1.
Upstream primer A1:
5’-GCGACCTGCTCCAGTCTCCAAAG-3’
Upstream primer A2:
5’-TTA
GGTACCGACGACGACGACAAGGCATCTCCGGGCTCT-3’
KpnI enteropeptidase restriction enzyme site
Downstream primer B1:
5’-GCT
GGATCCTGGAACAGCTTGGTGAGCA-3’
BamHI
The second step Nest-PCR amplification people GAD65DNA fragment
With human pancreas cDNA library (available from Clontech) is template, with high-fidelity Pfu archaeal dna polymerase amplification GAD65, earlier carry out pcr amplification and obtain elementary amplified production with above-mentioned elementary primer A1, B1, make template with elementary amplified production again, carry out pcr amplification with above-mentioned secondary primer A2, B1 and obtain people GAD65DNA fragment, electrophoresis detection, purifying also reclaims standby.
The 3rd step made up the genetic engineering bacterium that contains people GAD65 gene order
People GAD65 dna fragmentation with second step acquisition of two kinds of restriction enzymes double zyme cuttings, described two kinds of restriction enzymes are KpnI and BamHI, purifying reclaims small segment, with same two kinds of restriction enzymes double zyme cutting plasmid pET-32a (+), purifying reclaims big fragment, the small segment and the big fragment that connect above-mentioned recovery, must contain the segmental recombinant plasmid pET-32a-GAD65 of people GAD65DNA, pET-32a-GAD65 is transformed into bacillus coli DH 5 alpha with recombinant plasmid, make and contain people GAD65 gene order, it is people GAD65 DNA genetic engineering bacterium, entrust the biotech company of specialty to check order this genetic engineering bacterium, confirm that this genetic engineering bacterium contains complete recombinant human GAD65 gene fragment;
The 4th step made up the genetic engineering bacterium that efficiently expresses people GAD65
Genetic engineering bacterium extracting recombinant plasmid pET-32a-GAD65 from the 3rd step built is transformed into e. coli bl21 (DE3) or Rosetta-gami (DE3), the genetic engineering bacterium of the human glutamic acid decarboxylase 65 that obtains efficiently expressing.
Human pancreas cDNA library, plasmid pET-32a (+) that above-mentioned structure relates to, bacillus coli DH 5 alpha, BL21 (DE3), Rosetta-gami (DE3), restriction enzyme BamHI, KpnI and high-fidelity Pfu archaeal dna polymerase all can be buied from market.Entrusting the synthetic described primer sequence that gets and the biotech company of order-checking is Shanghai Hua Nuo Bioisystech Co., Ltd.
The purposes of the genetic engineering bacterium of efficiently expressing recombinant human GAD65 provided by the invention is for being used to produce soluble active Trx-human glutamic acid decarboxylase 65 fusion roteins that have.The present invention solves the problems of the technologies described above by following technical scheme, and the concrete operations step is as follows:
The first step liquid culture
The above-mentioned genetic engineering bacterium that efficiently expresses human glutamic acid decarboxylase 65 that builds is cultured to OD600nm=0.4～1.0 o'clock in the LB liquid nutrient medium, the adding final concentration is that the IPTG of 0.4～1.0mM carried out 16 ℃ of abduction deliverings of low temperature about 12 hours, produces and accumulate Trx-human glutamic acid decarboxylase 65 fusion roteins of solubility expression;
The second step purifying makes Trx-human glutamic acid decarboxylase 65 fusion roteins
Centrifugal collection thalline, with the resuspended thalline of damping fluid, after the carrying out ultrasonic bacteria breaking, centrifugal collection supernatant, carry out the His.Tag affinity chromatography, collecting Trx-human glutamic acid decarboxylase 65 fusion rotein components, is that the Millipore Amicon Ultra-15 ultrafiltration pipe of 5KD is concentrated into 2mg/mL with albumen with molecular weight cut-off;
The purposes of the genetic engineering bacterium of efficiently expressing recombinant human GAD65 provided by the invention is for being used to produce the soluble active recombinant human GAD65 that has.Concrete technical scheme is on the soluble basis with active Trx-human glutamic acid decarboxylase 65 fusion roteins of above-mentioned production, with enteropeptidase enzymolysis Trx-human glutamic acid decarboxylase 65 fusion roteins, 25 ℃ of following enzymolysis spend the night, termination reaction, carry out the His.Tag affinity chromatography once more, collection penetrates the peak, and lyophilize obtains RHGAD 65.
Compared with the prior art the present invention is than unusual effect:
(1) method by genetic engineering technique allows human glutamic acid decarboxylase 65 genes with the Trx amalgamation and expression, has improved the expression amount of soluble human L-Glutamic decarboxylase 65.
(2) by adding the His.Tag purification tag at the N of the dna sequence dna of human glutamic acid decarboxylase 65 genes end, as long as carry out the His.Tag affinity chromatography one time, just can from fermented liquid, obtain purer human glutamic acid decarboxylase 65 gene fusion albumen, purification step is easy, is easier to operation than the repeatedly chromatography of conventional art.
(3) as long as carry out the RHGAD 65 that primary enzymolysis just can not contained additional amino acid, purifying is very easy, the yield height.
(4) Trx of purifying-human glutamic acid decarboxylase 65 fusion roteins have biological activity, can select to remove Trx without the enteropeptidase enzymolysis and directly use, and have avoided the expensive shortcoming of enteropeptidase.
Thereby can be used for production RHGAD 65 or Trx-human glutamic acid decarboxylase 65 fusion roteins with genetic engineering bacterium provided by the invention, and the output height, purifying process is easy, and production cost is lower.
Description of drawings
Fig. 1 is the structure synoptic diagram of recombinant plasmid pET-32a-GAD65.
Fig. 2 is the SDS-PAGE electrophorogram of genetic engineering bacterium express recombinant human glutamic acid decarboxylase 65.
Fig. 3 is that the separation and purification and the enzyme of RHGAD 65 cut figure as a result.
Wherein 1 is the standard protein molecular mass; 2 is Trx-human glutamic acid decarboxylase 65 fusion roteins; 3 be Trx-human glutamic acid decarboxylase 65 fusion roteins through the enteropeptidase enzymolysis, 4 is the RHGAD 65 behind the purifying.
Fig. 4 is the active figure of detection of Trx-human glutamic acid decarboxylase 65 fusion rotein zymetologys.
Wherein 1 is L-Sodium Glutamate (L-MSG) and γ-An Jidingsuan (γ-GABA) contrast; 2 is the reaction solution of Trx-human glutamic acid decarboxylase 65 fusion rotein catalysis L-Sodium Glutamate decarboxylic reactions; 3 is the contrast of L-Sodium Glutamate decarboxylic reaction substrate solution.
Embodiment
Below in conjunction with accompanying drawing,, further specify the present invention by embodiment.The experimental technique of all unreceipted actual conditionses among specification sheets and the embodiment, condition is carried out routinely.
Embodiment 1: the preparation of genetic engineering bacterium
The first step design of primers
CDNA sequence according to known person GAD65, design specific Nest-PCR upstream primer A1, A2 and downstream primer B1, wherein introduce KpnI restriction enzyme digestion sites and enteropeptidase restriction enzyme site among the upstream primer A2, introduce the BamHI restriction enzyme digestion sites among the downstream primer B1.
Upstream primer A1:
5’-GCGACCTGCTCCAGTCTCCAAAG-3’
Upstream primer A2:
5’-TTA
GGTACCGACGACGACGACAAGGCATCTCCGGGCTCT-3’
KpnI enteropeptidase restriction enzyme site
Downstream primer B1:
5’-GCT
GGATCCTGGAACAGCTTGGTGAGCA-3’
BamHI
The second step Nest-PCR amplification people GAD65DNA fragment
With human pancreas cDNA library (available from Clontech) is template, with high-fidelity Pfu archaeal dna polymerase amplification GAD65, carries out pcr amplification and obtains elementary amplified production with above-mentioned elementary primer A1, B1 earlier, carries out pcr amplification by following condition: 94 ℃, and 40s; 54 ℃, 40s; 72 ℃, 2min circulates after 28 times 72 ℃ and extends 5min, obtains elementary amplified production.Make template with elementary amplified production again, carry out pcr amplification with above-mentioned secondary primer A2, B1 and obtain people GAD65DNA fragment, comprise in the PCR reaction system: 1 μ l template, it is elementary amplified production, 2 μ l 10X Pfu buffer, 1 μ l Pfu archaeal dna polymerase, 1 μ l dNTP (2.5mmol/L each), each 1 μ l of primer A2 and B1,12 μ l sterilized waters.The pcr amplification condition is: 94 ℃ of pre-sex change 5min, and 94 ℃ of 40s then, 55 ℃ of 40s, 72 ℃ of 2min circulate after 28 times 72 ℃ and extend 5min.Reaction product reclaims with DNA Gel Extraction Kit.Electrophoresis detection, purifying also reclaims standby.
The 3rd step made up the genetic engineering bacterium that contains people GAD65 gene order
People GAD65 dna fragmentation with the acquisition in second step of two kinds of restriction enzymes double zyme cuttings, described two kinds of restriction enzymes are KpnI and BamHI, purifying reclaims small segment, with same two kinds of restriction enzymes double zyme cutting plasmid pET-32a (+), purifying reclaims big fragment, the small segment and the big fragment that connect above-mentioned recovery, must contain the segmental recombinant plasmid pET-32a-GAD65 of people GAD65DNA, pET-32a-GAD65 is transformed into bacillus coli DH 5 alpha with recombinant plasmid, make and contain people GAD65 gene order, it is people GAD65DNA genetic engineering bacterium, entrust the biotech company of specialty to check order this genetic engineering bacterium, confirm that this genetic engineering bacterium contains complete recombinant human GAD65 gene fragment;
The 4th step made up the genetic engineering bacterium that efficiently expresses people GAD65
Extracting recombinant plasmid pET-32a-GAD65 from the 3rd genetic engineering bacterium that build of step is transformed among e. coli bl21 (DE3) or the Rosetta-gami (DE3) genetic engineering bacterium of the human glutamic acid decarboxylase 65 that obtains efficiently expressing.
A kind of application that efficiently expresses the genetic engineering bacterium of human glutamic acid decarboxylase 65 is promptly produced human glutamic acid decarboxylase 65 with described genetic engineering bacterium.Following affinity chromatography medium is Ni-NTA His.Bind or His.Bind resin, and available from Novagen company, following enteropeptidase is available from MERCK company.
The first step liquid culture
The genetic engineering bacterium that builds among the embodiment 1 is inoculated in 20mL contains in the LB liquid nutrient medium of 100mg/mL Amp, 37 ℃, 210rpm is cultured to OD600=0.6-1.0.5000rpm, 4 ℃, centrifugal 5min, the thalline of collecting precipitation, 4 ℃ of preservations are spent the night.Contained the resuspended thalline of LB substratum of 100mg/mL Amp with 20mL in second day, 4% volume is inoculated in 400mL and contains in the LB substratum of 100mg/mL Amp.37 ℃, 210rpm is cultured to OD600nm=0.4～1.0.Adding final concentration is 0.4～1.0mM IPTG, and 16 ℃～20 ℃ are carried out low temperature induction and express spend the night (about 12h).After will shaking bottle and placing on ice ice bath 5min, 6000rpm, 4 ℃, centrifugal 5-10min collects thalline.Add the resuspended thalline of 100mL NTA-0 or IDA-0Buffer, adding final concentration simultaneously is the N,O-Diacetylmuramidase of 0.2mg/mL and the PMSF of 1mM.Place 30min on ice, the ultrasonic disruption thalline, 12000rpm, 4 ℃, centrifugal 30min collects supernatant.
The second step purifying makes Trx-human glutamic acid decarboxylase 65 fusion roteins
Supernatant gets fusion rotein TrxA-GAD65 with affinity column Ni-NTA His.Bind or His.Bind resin separation purification.NTA-0 or IDA-0Buffer balance NTA/IDA-Resin with 5 times of resin volumes.Sample is added among the NTA/IDA-Resin, use the NTA/IDA-0 of 2 times of resin volumes respectively, NTA/IDA-20, NTA/IDA-40, NTA/IDA-60, NTA/IDA-80, NTA/IDA-100, NTA/IDA-200, the NTA/IDA-1000Buffer wash-out of 5 times of resin volumes, collect the elutriant of penetrating component and each component respectively, be used for SDS-PAGE analysing protein wash-out situation.Through SDS-PAGE analyze the elutriant molecular weight cut-off contain fusion rotein TrxA-GAD65 component be the Millipore Amicon Ultra-15 ultrafiltration pipe of 5KD-10KD with albumen desalination and concentrated, be concentrated into more than the 2mg/mL.Protein concentration is measured with the Lowry method.
Preparation human glutamic acid decarboxylase 65 on second basis that goes on foot of embodiment 3.Fusion rotein TrxA-GAD65 cuts with the enteropeptidase enzyme.Every milligram of fusion rotein adds the enteropeptidase of 1 unit, and 25 ℃, vibration, enzyme is cut about 16h.Enzyme is cut product affinity column Ni-NTA His.Bind or His.Bind resin isolation.Collection penetrates the elution peak of peak and NTA/IDA-0.Merge this two kinds of components, the MilliporeAmicon Ultra-15 ultrafiltration pipe of using 5KD-10KD is to GAD65 desalination and concentrated.Protein concentration is measured with the Lowry method.
TrxA-GAD65 or GAD65 the enzyme activity detect to adopt to measure by L-Sodium Glutamate (L-MSG) decarboxylation and generate the γ-An Jidingsuan (method of the amount of γ-GABA).10ul protein sample solution joins 100ul reaction substrate solution (50mM phosphoric acid buffer, pH7.2,1mM EDTA, 0.2mM 5 '-PLP, 20mML-MSG) in, mixing, 37 ℃ of water-baths, 30 minutes-1 hour, take out the 20ul reaction solution then, add the 20ul50mM borate buffer, pH9.0, add 4ul NBD-Cl (4mg/ml) again, 55 ℃ of water-baths 1 hour are got 3-4ul reaction solution point tlc silica gel plate and are carried out thin-layer chromatography, developing agent is a propyl carbinol, the mixing liquid of acetate and water, propyl carbinol wherein: acetate: water=4: 1.5: 1, the exhibition layer finishes rearmounted silica-gel plate under ultraviolet lamp, and L-MSG and γ-GABA place will show green fluorescence.Fluorescence is carried out photometric scanning can carry out quantitatively the γ-GABA that generates.
Embodiment 5 adopts human glutamic acid decarboxylase 65 fusion roteins or human glutamic acid decarboxylase 65 protein to carry out type i diabetes diagnostic detection (ELISA method)
Utilize human glutamic acid decarboxylase 65 fusion roteins or human glutamic acid decarboxylase 65 protein immunologic competences to detect.
Utilize the human glutamic acid decarboxylase 65 protein antibodies specificity bonded character among human glutamic acid decarboxylase 65 fusion roteins or human glutamic acid decarboxylase 65 albumen mass-energy and the type i diabetes patients serum, carry out according to the following steps:
A) human glutamic acid decarboxylase 65 fusion roteins or human glutamic acid decarboxylase 65 protein are cushioned liquid with the CBS bag and do different extent of dilution, and 100ul/ hole point sample is put into wet box with in enzyme mark hole, and 4 ℃ of bags are spent the night.
B) with the PBST washings (PBS pH7.4,0.05%Tween-20) 300ul/ hole vibration washing is three times, each 3-5min abandons most liquid.
C) seal up blocking solution (PBS pH7.4,0.05%Tween-20,2%BSA or 5% skim-milk) 200ul/ hole, 37 ℃ of wet boxes were hatched 1 hour.
D) repeating step b.
E) add type i diabetes patients serum 100ul/ hole, 37 ℃ of wet boxes were hatched 1 hour.
F) repeating step b.
G) add the mouse-anti human IgG of horseradish peroxidase-labeled, the 100ul/ hole, 37 ℃ of wet boxes were hatched 1 hour.
H) repeating step b.
I) add OPD colour developing liquid (OPD 0.5g/L is dissolved in 0.05mol/L citrate buffer solution pH5.0, contains 0.06%H2O2v/v, and is now with the current) 100ul/ hole, 25 ℃ of colour developing 30min.
J) 50ul/ hole stop buffer (2mol/L H2SO4) termination reaction, microplate reader 490nm measures each hole light absorption value.
A normal human serum contrast is set in the above ELISA test.Two multiple holes are done in each hole simultaneously, and each sample light absorption value adopts mean value to reduce error.
The sequencing result of recombinant plasmid pET-32a-GAD65 is as follows, can confirm that this genetic engineering bacterium contains complete recombinant human GAD65 gene fragment:
KpnI
1?tta?
ggt?acc?gac?gac?gac?gac?aag?gca?tct?ccg?ggc?tct?ggc
Asp?Asp?Asp?Asp?Lys?Ala?Ser?Pro?Gly?Ser?Gly
Enterokinas
43?ttt?tgg?tct?ttc?ggg?tcg?gaa?gat?ggc?tct?ggg?gat?tcc?gag
Phe?Trp?Ser?Phe?Gly?Ser?Glu?Asp?Gly?Ser?Gly?Asp?Ser?Glu
85?aat?ccc?ggc?aca?gcg?cga?gcc?tgg?tgc?caa?gtg?gct?cag?aag
Asn?Pro?Gly?Thr?Ala?Arg?Ala?Trp?Cys?Gln?Val?Ala?Gln?Lys
127?ttc?acg?ggc?ggc?atc?gga?aac?aaa?ctg?tgc?gcc?ctg?ctc?tac
Phe?Thr?Gly?Gly?Ile?Gly?Asn?Lys?Leu?Cys?Ala?Leu?Leu?Tyr
169?gga?gac?gcc?gag?aag?ccg?gcg?gag?agc?ggc?ggg?agc?caa?ccc
Gly?Asp?Ala?Glu?Lys?Pro?Ala?Glu?Ser?Gly?Gly?Ser?Gln?Pro
211?ccg?cgg?gcc?gcc?gcc?cgg?aag?gcc?gcc?tgc?gcc?tgc?gac?cag
Pro?Arg?Ala?Ala?Ala?Arg?Lys?Ala?Ala?Cys?Ala?Cys?Asp?Gln
253?aag?ccc?tgc?agc?tgc?tcc?aaa?gtg?gat?gtc?aac?tac?gcg?ttt
Lys?Pro?Cys?Ser?Cys?Ser?Lys?Val?Asp?Val?Asn?Tyr?Ala?Phe
295?ctc?cat?gca?aca?gac?ctg?ctg?ccg?gcg?tgt?gat?gga?gaa?agg
Leu?His?Ala?Thr?Asp?Leu?Leu?Pro?Ala?Cys?Asp?Gly?Glu?Arg
337?ccc?act?ttg?gcg?ttt?ctg?caa?gat?gtt?atg?aac?att?tta?ctt
Pro?Thr?Leu?Ala?Phe?Leu?Gln?Asp?Val?Met?Asn?Ile?Leu?Leu
379?cag?tat?gtg?gtg?aaa?agt?ttc?gat?aga?tca?acc?aaa?gtg?att
Gln?Tyr?Val?Val?Lys?Ser?Phe?Asp?Arg?Ser?Thr?Lys?Val?Ile
421?gat?ttc?cat?tat?cct?aat?gag?ctt?ctc?caa?gaa?tat?aat?tgg
Asp?Phe?His?Tyr?Pro?Asn?Glu?Leu?Leu?Gln?Glu?Tyr?Asn?Trp
463?gaa?ttg?gca?gac?caa?cca?caa?aat?ttg?gag?gaa?att?ttg?atg
Glu?Leu?Ala?Asp?Gln?Pro?Gln?Asn?Leu?Glu?Glu?Ile?Leu?Met
505?cat?tgc?caa?aca?act?cta?aaa?tat?gca?att?aaa?aca?ggg?cat
His?Cys?Gln?Thr?Thr?Leu?Lys?Tyr?Ala?Ile?Lys?Thr?Gly?His
547?cct?aga?tac?ttc?aat?caa?ctt?tct?act?ggt?ttg?gat?atg?gtt
Pro?Arg?Tyr?Phe?Asn?Gln?Leu?Ser?Thr?Gly?Leu?Asp?Met?Val
589?gga?tta?gca?gca?gac?tgg?ctg?aca?tca?aca?gca?aat?act?aac
Gly?Leu?Ala?Ala?Asp?Trp?Leu?Thr?Ser?Thr?Ala?Asn?Thr?Asn
631?atg?ttc?acc?tat?gaa?att?gct?cca?gta?ttt?gtg?ctt?ttg?gaa
Met?Phe?Thr?Tyr?Glu?Ile?Ala?Pro?Val?Phe?Val?Leu?Leu?Glu
673?tat?gtc?aca?cta?aag?aaa?atg?aga?gaa?atc?att?ggc?tgg?cca
Tyr?Val?Thr?Leu?Lys?Lys?Met?Arg?Glu?Ile?Ile?Gly?Trp?Pro
715?ggg?ggc?tct?ggc?gat?ggg?ata?ttt?tct?ccc?ggt?ggc?gcc?ata
Gly?Gly?Ser?Gly?Asp?Gly?Ile?Phe?Ser?Pro?Gly?Gly?Ala?Ile
757?tct?aac?atg?tat?gcc?atg?atg?atc?gca?cgc?ttt?aag?atg?ttc
Ser?Asn?Met?Tyr?Ala?Met?Met?Ile?Ala?Arg?Phe?Lys?Met?Phe
799?cca?gaa?gtc?aag?gag?aaa?gga?atg?gct?gct?ctt?ccc?agg?ctc
Pro?Glu?Val?Lys?Glu?Lys?Gly?Met?Ala?Ala?Leu?Pro?Arg?Leu
841?att?gcc?ttc?acg?tct?gaa?cat?agt?cat?ttt?tct?ctc?aag?aag
Ile?Ala?Phe?Thr?Ser?Glu?His?Ser?His?Phe?Ser?Leu?Lys?Lys
883?gga?gct?gca?gcc?tta?ggg?att?gga?aca?gac?agc?gtg?att?ctg
Gly?Ala?Ala?Ala?Leu?Gly?Ile?Gly?Thr?Asp?Ser?Val?Ile?Leu
925?att?aaa?tgt?gat?gag?aga?ggg?aaa?atg?att?cca?tct?gat?ctt
Ile?Lys?Cys?Asp?Glu?Arg?Gly?Lys?Met?Ile?Pro?Ser?Asp?Leu
967?gaa?aga?agg?att?ctt?gaa?gcc?aaa?cag?aaa?ggg?ttt?gtt?cct
Glu?Arg?Arg?Ile?Leu?Glu?Ala?Lys?Gln?Lys?Gly?Phe?Val?Pro
1009?ttc?ctc?gtg?agt?gcc?aca?gct?gga?acc?acc?gtg?tac?gga?gca
Phe?Leu?Val?Ser?Ala?Thr?Ala?Gly?Thr?Thr?Val?Tyr?Gly?Ala
1051?ttt?gac?ccc?ctc?tta?gct?gtc?gct?gac?att?tgc?aaa?aag?tat
Phe?Asp?Pro?Leu?Leu?Ala?Val?Ala?Asp?Ile?Cys?Lys?Lys?Tyr
1093?aag?atc?tgg?atg?cat?gtg?gat?gca?gct?tgg?ggt?ggg?gga?tta
Lys?Ile?Trp?Met?His?Val?Asp?Ala?Ala?Trp?Gly?Gly?Gly?Leu
1135?ctg?atg?tcc?cga?aaa?cac?aag?tgg?aaa?ctg?agt?ggc?gtg?gag
Leu?Met?Ser?Arg?Lys?His?Lys?Trp?Lys?Leu?Ser?Gly?Val?Glu
1177?agg?gcc?aac?tct?gtg?acg?tgg?aat?cca?cac?aag?atg?atg?gga
Arg?Ala?Asn?Ser?Val?Thr?Trp?Asn?Pro?His?Lys?Met?Met?Gly
1219?gtc?cct?ttg?cag?tgc?tct?gct?ctc?ctg?gtt?aga?gaa?gag?gga
Val?Pro?Leu?Gln?Cys?Ser?Ala?Leu?Leu?Val?Arg?Glu?Glu?Gly
1261?ttg?atg?cag?aat?tgc?aac?caa?atg?cat?gcc?tcc?tac?ctc?ttt
Leu?Met?Gln?Asn?Cys?Asn?Gln?Met?His?Ala?Ser?Tyr?Leu?Phe
1303?cag?caa?gat?aaa?cat?tat?gac?ctg?tcc?tat?gac?act?gga?gac
Gln?Gln?Asp?Lys?His?Tyr?Asp?Leu?Ser?Tyr?Asp?Thr?Gly?Asp
1345?aag?gcc?tta?cag?tgc?gga?cgc?cac?gtt?gat?gtt?ttt?aaa?cta
Lys?Ala?Leu?Gln?Cys?Gly?Arg?His?Val?Asp?Val?Phe?Lys?Leu
1387?tgg?ctg?atg?tgg?agg?gca?aag?ggg?act?acc?ggg?ttt?gaa?gcg
Trp?Leu?Met?Trp?Arg?Ala?Lys?Gly?Thr?Thr?Gly?Phe?Glu?Ala
1429?cat?gtt?gat?aaa?tgt?ttg?gag?ttg?gca?gag?tat?tta?tac?aac
His?Val?Asp?Lys?Cys?Leu?Glu?Leu?Ala?Glu?Tyr?Leu?Tyr?Asn
1471?atc?ata?aaa?aac?cga?gaa?gga?tat?gag?atg?gtg?ttt?gat?ggg
Ile?Ile?Lys?Asn?Arg?Glu?Gly?Tyr?Glu?Met?Val?Phe?Asp?Gly
1513?aag?cct?cag?cac?aca?aat?gtc?tgc?ttc?tgg?tac?att?cct?cca
Lys?Pro?Gln?His?Thr?Asn?Val?Cvs?Phe?Trp?Tyr?Ile?Pro?Pro
1555?agc?ttg?cgt?act?ctg?gaa?gac?aat?gaa?gag?aga?atg?agt?cgc
Ser?Leu?Arg?Thr?Leu?Glu?Asp?Asn?Glu?Glu?Arg?Met?Ser?Arg
1597?ctc?tcg?aag?gtg?gct?cca?gtg?att?aaa?gcc?aga?atg?atg?gag
Leu?Ser?Lys?Val?Ala?Pro?Val?Ile?Lys?Ala?Arg?Met?Met?Glu
1639?tat?gga?acc?aca?atg?gtc?agc?tac?caa?ccc?ttg?gga?gac?aag
Tyr?Gly?Thr?Thr?Met?Val?Ser?Tyr?Gln?Pro?Leu?Gly?Asp?Lys
1681?gtc?aat?ttc?ttc?cgc?atg?gtc?atc?tca?aac?cca?gcg?gca?act
Val?Asn?Phe?Phe?Arg?Met?Val?Ile?Ser?Asn?Pro?Ala?Ala?Thr
1723?cac?caa?gac?att?gac?ttc?ctg?att?gaa?gaa?ata?gaa?cgc?ctt
His?Gln?Asp?Ile?Asp?Phe?Leu?Ile?Glu?Glu?Ile?GLu?Arg?Leu
1765?gga?caa?gat?tta?taa?taa?cct?tgc?tca?cca?agc?tgt?tcc?a
gg
Gly?Gln?Asp?Leu ---?---
BamHI
1807?
atc?cga?att
Claims (9)
1. genetic engineering bacterium, constitute by recombinant plasmid and host bacterium, it is characterized in that recombinant plasmid refers to the plasmid pET-32a (+) that contains the GAD65 gene, the host bacterium refers to any one among bacillus coli DH 5 alpha, BL21 (DE3) or the Rosetta-gami (DE3).
2. genetic engineering bacterium as claimed in claim 1 is characterized in that: recombinant plasmid is pET-32a (+)-P
T7-TrxA-6 * His-GAD65, wherein, P
T7It is the promotor of plasmid pET-32a (+); TrxA is a Trx; 6 * His is the Histidine purification tag.
3. the preparation method of a genetic engineering bacterium, it is characterized in that containing the enteropeptidase restriction enzyme site by the PCR primer of design, people GAD65 gene fragment with pcr amplification, through the restricted type endonuclease digestion, be cloned among the expression plasmid pET-32a (+), be built into recombinant plasmid pET-32a-GAD65, be transformed into then in the host bacterium, the genetic engineering bacterium that makes.
4. the preparation method of genetic engineering bacterium as claimed in claim 3 is characterized in that comprising the steps:
The first step primer design is with synthetic
CDNA sequence according to people GAD65, design specific Nest-PCR upstream primer A1, A2 and downstream primer B1, wherein introduce KpnI restriction enzyme digestion sites and enteropeptidase restriction enzyme site among the upstream primer A2, introduce the BamHI restriction enzyme digestion sites among the downstream primer B1;
Upstream primer A1:
5’-GCGACCTGCTCCAGTCTCCAAAG-3’
Upstream primer A2:
5’-TTA
GCATCTCCGGGCTCT-3’
KpnI enteropeptidase restriction enzyme site
Downstream primer B1:
5’-GCT
TGGAACAGCTTGGTGAGCA-3’
BamHI
The second step Nest-PCR amplification people GAD65DNA fragment
With human pancreas cDNA library is template, with high-fidelity Pfu archaeal dna polymerase amplification GAD65, earlier carry out pcr amplification and obtain elementary amplified production with elementary primer A1, B1, make template with elementary amplified production again, carry out pcr amplification with secondary primer A2, B1 and obtain people GAD65 dna fragmentation, purifying also reclaims standby;
The 3rd step made up the genetic engineering bacterium that contains people GAD65 gene order
People GAD65 dna fragmentation with two kinds of restriction enzyme KpnI and second step acquisition of BamHI double digestion, purifying reclaims small segment, use KpnI and BamHI double digestion plasmid pET-32a (+) again, purifying reclaims big fragment, the small segment and the big fragment that connect above-mentioned recovery, the recombinant plasmid pET-32a-GAD65 that must contain people GAD65 dna fragmentation is transformed into the host bacterium with recombinant plasmid pET-32a-GAD65 and makes people GAD65 DNA genetic engineering bacterium.
5. as the preparation method of claim 3 or 4 described genetic engineering bacteriums, it is characterized in that the host bacterium is a bacillus coli DH 5 alpha, any one among BL21 (DE3) or the Rosetta-gami (DE3).
6. the application of genetic engineering bacterium in preparation Trx-human glutamic acid decarboxylase 65 fusion roteins.
7. the application of genetic engineering bacterium as claimed in claim 6 in preparation Trx-human glutamic acid decarboxylase 65 fusion roteins is characterized in that comprising the steps:
The first step liquid culture
The genetic engineering bacterium for preparing is cultured to OD600nm=0.4～1.0 o'clock in the LB liquid nutrient medium, the IPTG that adds final concentration and be 0.4～1.0mM carries out low temperature induction to express about 12 hours, produced and Trx-human glutamic acid decarboxylase 65 fusion roteins of accumulation solubility expression;
The second step purifying makes Trx-human glutamic acid decarboxylase 65 fusion roteins
Centrifugal collection thalline, with the resuspended thalline of damping fluid, after the carrying out ultrasonic bacteria breaking, centrifugal collection supernatant, carry out the His.Tag affinity chromatography, collecting Trx-human glutamic acid decarboxylase 65 fusion rotein components, is that the Millipore Amicon Ultra-15 ultrafiltration pipe of 5KD is concentrated into 2mg/mL with albumen with molecular weight cut-off;
8. the application of genetic engineering bacterium in preparation RHGAD 65.
9. the application of genetic engineering bacterium as claimed in claim 8 in preparation RHGAD 65, it is characterized in that Trx-human glutamic acid decarboxylase 65 fusion roteins enteropeptidase enzymolysis Trx-human glutamic acid decarboxylase 65 fusion roteins, 25 ℃ of following enzymolysis spend the night, termination reaction, carry out the His.Tag affinity chromatography once more, collection penetrates the peak, and lyophilize obtains RHGAD 65.
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| CN106399342A (en) * | 2016-11-14 | 2017-02-15 | 天津大学 | Recombinant plasmid, recombinant bacterium, construction method of recombinant plasmid and application of recombinant plasmid |
| CN106399342B (en) * | 2016-11-14 | 2019-12-31 | 天津大学 | Recombinant plasmid and recombinant engineering bacterium as well as construction method and application thereof |
| CN111378611A (en) * | 2018-12-29 | 2020-07-07 | 杭州唯铂莱生物科技有限公司 | Glutamic acid decarboxylase recombinant bacterium and construction method and application thereof |
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