CN104726497A - Expression and purification method for paraoxonase 1 gene - Google Patents

Abstract

Belonging to the technical field of genetic engineering, the invention discloses an expression and purification method for paraoxonase 1 gene. The method includes: firstly amplifying paraoxonase 1 gene, performing connection to a transfer vector pFastBacTM I to construct a recombinant transfer vector, transforming an Escherichia coli DH10Bac competent cell to obtain recombinant baculovirus DNA containing the paraoxonase 1 gene, transfecting a Bombyx mori BmN cell with the recombinant baculovirus DNA, carrying out replication and assembly to obtain a recombinant virus containing the paraoxonase 1 gene, then inoculating Bombyx mori fifth instar larva with the recombinant virus, taking the Bombyx mori blood after incidence, carrying out centrifugal filtration, impurity removing and concentration, then performing Cibacron Blue affinity chromatography and antibody affinity chromatography to purify the Bombyx mori blood so as to obtain paraoxonase 1 protein. The method provided by the invention realizes high-efficiency expression of protein, the structure is close to natural protein, and the problems of low content of natural PON1 and difficult purification are solved.

Description

The expression and purification method of PON1 gene

Technical field

The invention belongs to genetically engineered and protein engineering techniques field, be specifically related to the expression and purification method of PON1 gene.

Background technology

PON1 (paraoxonase1, PON1) in research organophosphate poisoning prevention and therapy, there is vital role, but current natural PON1 is difficult to obtain, although utilize the PON1 of escherichia coli expression cheap and with short production cycle, but the albumen obtained is because lacking glycosylation and phosphorylation modification, the problems such as there is immunological rejection during application in vivo, the transformation period is short, easy degraded.

Although also have the report being given expression to the activated PON1 gene of tool by gene engineering method at present, fail to obtain the recombinant protein of purifying, also just there is no the expression and purification method of fairly perfect PON1.

Summary of the invention

In order to solve the problem of expression, purifying tool activated PON1 gene difficulty in prior art, the invention provides a kind of expression and purification method of PON1 gene.

The expression and purification method of PON1 gene of the present invention, step is as follows:

(1) increase PON1 gene, is connected to transfer vector pFastBac ^tMrecombinant transfer vector is built on 1, recombinant transfer vector transformation of E. coli DH10Bac competent cell, obtain the recombinant baculovirus DNA containing PON1 gene, make it be assembled into the recombinant virus containing PON1 gene in time multiplexed cell system recombinant baculovirus DNA transfection Bombyx noriN cell;

(2) the recombinant virus inoculation silkworm five-age larva of step (1), gets silkworm blood after morbidity, purifies silkworm blood through centrifuging removal of impurities and after concentrated, acquisition PON1 albumen by ciba blue affinity chromatography and antibody affinity chromatography.

Preferably, in the expression and purification method of above-mentioned PON1 gene, the primer nucleotide sequences that the described amplification of step (1) uses is as shown in SEQ ID NO:1 ~ 2.

Preferably, in the expression and purification side of above-mentioned PON1 gene, step (2) described centrifugal be with centrifugal 30 min of the rotating speed of 10000 rpm under 4 DEG C of conditions; Described filtering and impurity removing is again through 0.45 μm of filter membrane after employing 9 layers of hospital gauze filter; Described concentrating is carried out with 10 kD super filter tubes.

Preferably, in the expression and purification method of above-mentioned PON1 gene, the stationary phase that the described ciba blue affinity chromatography of step (2) uses is ciba blue agarose-3GA3000-CL.

Preferably, in the expression and purification method of above-mentioned PON1 gene, step (2) described antibody affinity chromatography antibody used gets blood after PON1 gene four immunize New Zealand male rabbits by prokaryotic expression then to be obtained by ProteinA purifying.

Beneficial effect of the present invention: by design primer, construction of recombinant virus vBm- pON1, inoculated silkworm five-age larva, make it express target protein PON1, obtained the higher PON1 of purity by ciba blue affinity chromatography and antibody affinity chromatography purifying.It is low that this method solves natural PON1 content, the rare problem of purifying, lays the foundation for studying PON1 function later.

Accompanying drawing explanation

Fig. 1 is recombinant transfer vector pFastBac ^tM1- pON1double digestion-PCR identify figure M:DNA ladder marker; 1: double digestion product; 2:PCR product.

Fig. 2 is restructuring Bacmid- pON1pCR qualification figure; M.DNA ladder marker, 1.PON1 F/PON1R PCR primer, 2. PON1 F/M13R PCR product, 3. M13F/PON1R PCR product, 4. M13F/M13R PCR primer.

Fig. 3 is recombinant baculovirus BmNPV- pON1the SDS-PAGE figure of expression product in silkworm five-age larva, M. protein low molecule quality status stamp, 1. inoculates the silkworm blood of wild virus, 2. inoculates the silkworm blood of recombinant virus.

Fig. 4 is recombinant baculovirus BmNPV- pON1in silkworm five-age larva, the Western blotting of expression product identifies figure, M. protein low molecule quality status stamp, 1 '. the silkworm blood of inoculation wild virus, 2 '. and the silkworm blood of inoculation recombinant virus.

Fig. 5 is the SDS-PAGE qualification figure of purifying silkworm blood, M.: protein low molecule quality status stamp, 1: the silkworm blood of inoculation wild virus, 2: the silkworm blood former state of inoculation recombinant virus, 3: the silkworm blood of ciba blue affinity column preliminary purification, 4: the silkworm blood that antibody affinity chromatography is further purified.

Fig. 6 is the Western blotting qualification figure of purifying silkworm blood, M.: protein low molecule quality status stamp, 1 ': the silkworm blood of inoculation wild virus, 2 ': the silkworm blood former state of inoculation recombinant virus, 3 ': the silkworm blood of ciba blue affinity column preliminary purification, 4 ': the silkworm blood that antibody affinity chromatography is further purified.

Embodiment

Below in conjunction with specific embodiment, the invention will be further described, and to make those skilled in the art the present invention may be better understood and can be implemented, but illustrated embodiment is not as a limitation of the invention.

Biomaterial and reagent source:

PFastBac ^tM1 carrier purchased from Invitrogen company, restriction enzyme ecoRi He xhoi purchased from Fermentas company, ligase enzyme Ligation high is purchased from Fermentas company, e. coli tg1 competent cell and intestinal bacteria DH10Bac competent cell are purchased from Fermentas company, Sf-900 II SFM (1 ×) serum free medium is purchased from GIBCO company of the U.S., and foetal calf serum (FBS) is purchased from Austrian PAA company.Bombyx noriN cell in the present embodiment and silkworm five-age larva are so kind as to give by biochemical institute teacher Wu Xiangfu in Shanghai, and the public can buy similar commodity in market, and present invention applicant also can provide, and can not affect the realization of goal of the invention.

Embodiment 1: recombinant transfer vector pFastBac ^tM1- pON1structure

According to PON1 genetic characteristics, design primers F and R, with silkworm total serum IgE for template carries out reverse transcription synthesis cDNA, take cDNA as template, F and R is primer, carries out pcr amplification.

Upstream primer F:5 ^'-CCG gAATTCaTGGCTAAACTGACAGCG-3 ' (SEQ ID NO:1), wherein underscore is ecor I restriction enzyme site;

Downstream primer R:5 ^{' _}cCG cTCGAGcTACAGCTCACAGTAAAG-3 ' (SEQ ID NO:2), underscore is xhoi restriction enzyme site.

Amplification program is: 95 DEG C of denaturation 5min, 95 DEG C of sex change 45s, 59 DEG C of annealing 30s, and 72 DEG C extend 1min, circulate 30 times; Last 72 DEG C extend 8min, reclaim product, obtain PON1 (PON1) gene.

Will pON1 gene and pFastBac ^tM1 carrier is used respectively ecoRi He xhoi carries out double digestion simultaneously, and agarose gel electrophoresis reclaims digestion products; The PON1 gene fragment Ligation high ligase enzyme of recovery is connected to the pFastBac cut through enzyme ^tMon 1 carrier, PON1 gene is positioned under polyhedron promoter control, builds transfer vector pFastBac ^tM1-PON1, transformation of E. coli TG1 competent cell, ordinary method screening positive clone, this recombinant transfer vector is carried out PCR and double digestion, product carries out agarose gel electrophoresis to be identified, as shown in Figure 1, two bright bands appear to result in No. 1 swimming lane double digestion product, wherein one is the PON1 gene fragment of 1065bp, and another is fragment after plasmid enzyme restriction; Bright band in No. 2 swimming lanes is the PCR primer of the PON1 gene fragment of 1065bp, agarose gel electrophoresis preliminary evaluation plasmid construction success, then send to order-checking, through order-checking know that its sequence is as shown in SEQ ID NO:3, goal gene successful clone to pFastBac ^tMon 1 carrier.

Embodiment 2: the structure of silkworm with recombinant baculovirus vBm-PON1

(1) acquisition of recombinant baculovirus DNA (Bacmid-PON1)

The recombinant transfer vector pFastBac that embodiment 1 is built ^tM1-PON1 is transformed in intestinal bacteria DH10Bac competent cell, positive colony is obtained by blue hickie screening, respectively with M13 upstream and downstream primer, PON1 upstream and downstream, M13 downstream primer and PON1 upstream primer and M13 upstream primer and PON1 downstream primer four groups of primer pairs, carry out PCR qualification, wherein M13F:5 '-CCCAGTCACGACGTTGTAAAACG-3 ' (SEQ ID NO:4); M13R:5 '-AGCGGATAACAATTTTCACACAGG-3 ' (SEQ ID NO:5), the PCR primer that takes a morsel carries out agarose gel electrophoresis electrophoresis, and result as shown in Figure 2, shows to obtain correct Bacmid-PON1 positive colony.

(2) recombinant baculovirus DNA (Bacmid-PON1) transfection Bombyx noriN cell

A. in advance Bombyx noriN cell pipettor good for growth conditions is got 1mL and is laid in minicell culture dish (specification is 3.5cm) by 8 ~ 12h.

B. get 10 μ L Bacmid-PON1 and 8 μ L liposomes in sterilized EP pipe, add Sf-900 II SFM (1 ×) serum free medium of 100 μ L, incubated at room is about 20min, obtains Bacmid-PON1/ liposome complex.

C. add 2mL Sf-900 II SFM (1 ×) serum free medium after Bombyx noriN cell in good condition for adherent growth in step a being discarded substratum to wash twice, to remove foetal calf serum residual in former substratum.

D. add fresh Sf-900 II SFM (1 ×) serum free medium 1mL, then add Bacmid-PON1/ liposome complex 100 μ L, rock culture dish gently and mixture is evenly distributed; Hatch about 6h in 28 DEG C of cell culture incubators in this substratum, add 10 μ L foetal calf serums (FBS) continuation cultivations.

E., after 28 DEG C of cultivation 3 ~ 5d, (cell free is in substratum for typical disease symptom: cell becomes circle by spindle shape, kernel increase for the onset state of basis of microscopic observation cell; And normal cell arranges tight and is fusiformis, nucleus is obvious, and cell attachment growth is obvious).Cell morbidity shows restructuring Bacmid Successful transfection Bombyx noriN cell, and successfully carries out copying, assembling formation recombinant virus vBm-PON1 in cell.

Embodiment 3 silkworm with recombinant baculovirus vBm-PON1 inoculates SDS-PAGE and the Western blotting Analysis and Identification of silkworm five-age larva

Needle punching inoculation silkworm (mountain valley with clumps of trees and bamboo pine × bright moon) five-age larva after silkworm with recombinant baculovirus vBm-PON1 increases, collects silkworm hemolymph and carries out next step experiment or frozen in-80 DEG C of refrigerators after its morbidity.

The silkworm larva hemolymph treatment process of morbidity: by hemolymph after 9 layers of hospital gauze filter, through the centrifugal 10min of 2000rpm, collects supernatant, precipitates resuspended with the physiological saline of 0.9% for 4 DEG C.Carry out SDS-PAGE and Western blotting to the silkworm hemolymph after process to analyze.As shown in Figure 3 and Figure 4, there is specific band near 43kD in display supernatant, silkworm with recombinant baculovirus successful expression in silkworm five-age larva be described in result.

Embodiment 4 purifies silkworm blood by ciba blue affinity chromatography and antibody affinity chromatography

By centrifugal for silkworm blood 30 min(4 DEG C, 10000 rpm), get after supernatant crosses 9 layers of hospital gauze and cross 0.45 μm of filter membrane with removal of impurities, with 10 kD super filter tubes, it is concentrated.Main and the high-density lipoprotein (HDL) (HDL) of paraoxonase (PON) in serum combines, and ciba blue agarose-3GA3000-CL has specific adsorption to serum lipoprotein.Therefore select it as affinity chromatography medium, affinity chromatography preliminary purification is carried out to above-mentioned concentrated silkworm blood, and then is further purified silkworm blood with antibody affinity chromatography.Concrete purification process is as follows:

1. the concentration of silkworm hemolymph

(1) the silkworm blood taking out preservation from-80 DEG C of refrigerators is placed on ice to after dissolving completely, centrifugal 30 min(4 DEG C, 10000 rpm), supernatant is crossed 9 layers of hospital gauze to remove lipid material.

(2) filtrate uses 0.45 μm of membrane filtration again, collects filtrate.

(3) with 10 kD super filter tubes, filtrate is concentrated.

2. ciba blue affinitive layer purification

Main and the high-density lipoprotein (HDL) (HDL) of paraoxonase (PON) in serum combines, and ciba blue agarose-3GA3000-CL has specific adsorption to serum lipoprotein.Therefore select it as affinity chromatography medium, affinity chromatography is carried out to the object zymoprotein in serum.Concrete steps are as follows:

(1) get concentrated silkworm blood 10 mL, add the level pad of 30 mL, then mix after adding 7 mL ciba blue agaroses, 4 DEG C of stirrings are spent the night, and the albumen in serum is fully combined with ciba blue.

(2) post is filled, with 30 mL level pad continuous flushings, to wash the albumen (utilizing the salting out of balance liquid) be not attached on filler off.

(3) chromatography column desalination is rinsed with the lavation buffer solution of 30 mL.

(4) carry out wash-out with 30 mL elution buffers, collect elutriant, and concentrated desalination (PBS with pH 7.4).

3. antibody affinity chromatography purifying

(1) the PON1 gene that embodiment 1 obtains obtains albumen by prokaryotic expression, gets blood and then obtains antibody by ProteinA purifying, the purified rear Dispersal risk affinity column of antibody after gained albumen four immunize New Zealand male rabbits.

(2) the silkworm blood sample antibody affinity chromatography after concentrated is carried out purifying.

(3) elutriant of collection is concentrated, and detect protein concentration in sample (BCA protein quantification test kit).

4. two kinds of chromatographic eluate are concentrated respectively, SDS-PAGE and Western blotting is carried out to it and analyzes.Result as shown in Figure 5 and Figure 6, shows by twice chromatographic, obtains purer silkworm blood PON1, illustrates that this purification process is very successful.PON1 protein amino acid sequence is as shown in SEQ ID NO:6.

The above embodiment is only that protection scope of the present invention is not limited thereto in order to absolutely prove the preferred embodiment that the present invention lifts.The equivalent alternative or conversion that those skilled in the art do on basis of the present invention, all within protection scope of the present invention.Protection scope of the present invention is as the criterion with claims.

SEQUENCE LISTING

 

<110> Tianjin Yaoyu Biotechnology Co., Ltd.

 

The expression and purification method of <120> PON1 gene

 

<130>

 

<160> 6

 

<170> PatentIn version 3.3

 

<210> 1

<211> 27

<212> DNA

<213> artificial sequence

 

<400> 1

ccggaattca tggctaaact gacagcg 27

 

 

<210> 2

<211> 27

<212> DNA

<213> artificial sequence

 

<400> 2

ccgctcgagc tacagctcac agtaaag 27

 

 

<210> 3

<211> 1065

<212> DNA

<213> artificial sequence

 

<400> 3

atggctaaac tgacagcgct cacactcttg gggctgggat tggcactctt cgatggacag 60

 

aagtcttctt tccaaacacg atttaatgtt caccgtgaag taactccagt ggaacttcct 120

 

aactgtaatt tagttaaagg ggttgacaat ggttctgaag acttggaaat actgcccaat 180

 

ggactggctt tcatcagctc cggattaaag tatcctggaa taatgagctt tgaccctgat 240

 

aagtctggaa agatacttct aatggacctg aatgaggaag acccagtagt gttggaactg 300

 

ggcattactg gaaatacatt ggatatatct tcatttaacc ctcatgggat tagcacattc 360

 

acagatgaag ataacactgt gtacctactg gtggtaaacc atccagactc ctcgtccacc 420

 

gtggaggtgt ttaaatttca agaagaagaa aaatcacttt tgcatctgaa aaccatcaga 480

 

cacaagcttc tgcctagtgt gaatgacatt gtcgctgtgg gacctgaaca cttttatgcc 540

 

acaaatgatc actattttgc tgacccttac ttaaaatcct gggaaatgca tttgggatta 600

 

gcgtggtcat ttgttactta ttatagtccc aatgatgttc gagtagtggc agaaggattt 660

 

gattttgcta acggaatcaa catctcacca gacggcaagt atgtctatat agctgagttg 720

 

ctggctcata agatccatgt gtatgaaaag cacgctaatt ggactttaac tccattgaag 780

 

tccctcgact ttgacaccct tgtggataac atctctgtgg atcctgtgac aggggacctc 840

 

tgggtgggat gccatcccaa cggaatgcga atcttctact atgacccaaa gaatcctccc 900

 

ggctcagagg tgcttcgaat ccaggacatt ttatccgaag agcccaaagt gacagtggtt 960

 

tatgcagaaa atggcactgt gttacagggc agcacggtgg ccgctgtgta caaagggaaa 1020

 

ctgctgattg gcacagtgtt tcacaaagct ctttactgtg agctg 1065

 

 

<210> 4

<211> 23

<212> DNA

<213> artificial sequence

 

<400> 4

cccagtcacg acgttgtaaa acg 23

 

 

<210> 5

<211> 24

<212> DNA

<213> artificial sequence

 

<400> 5

agcggataac aattttcaca cagg 24

 

 

<210> 6

<211> 355

<212> PRT

<213> artificial sequence

 

<400> 6

 

Met Ala Lys Leu Thr Ala Leu Thr Leu Leu Gly Leu Gly Leu Ala Leu

1 5 10 15

 

 

Phe Asp Gly Gln Lys Ser Ser Phe Gln Thr Arg Phe Asn Val His Arg

20 25 30

 

 

Glu Val Thr Pro Val Glu Leu Pro Asn Cys Asn Leu Val Lys Gly Val

35 40 45

 

 

Asp Asn Gly Ser Glu Asp Leu Glu Ile Leu Pro Asn Gly Leu Ala Phe

50 55 60

 

 

Ile Ser Ser Gly Leu Lys Tyr Pro Gly Ile Met Ser Phe Asp Pro Asp

65 70 75 80

 

 

Lys Ser Gly Lys Ile Leu Leu Met Asp Leu Asn Glu Glu Asp Pro Val

85 90 95

 

 

Val Leu Glu Leu Gly Ile Thr Gly Asn Thr Leu Asp Ile Ser Ser Phe

100 105 110

 

 

Asn Pro His Gly Ile Ser Thr Phe Thr Asp Glu Asp Asn Thr Val Tyr

115 120 125

 

 

Leu Leu Val Val Asn His Pro Asp Ser Ser Ser Thr Val Glu Val Phe

130 135 140

 

 

Lys Phe Gln Glu Glu Glu Lys Ser Leu Leu His Leu Lys Thr Ile Arg

145 150 155 160

 

 

His Lys Leu Leu Pro Ser Val Asn Asp Ile Val Ala Val Gly Pro Glu

165 170 175

 

 

His Phe Tyr Ala Thr Asn Asp His Tyr Phe Ala Asp Pro Tyr Leu Lys

180 185 190

 

 

Ser Trp Glu Met His Leu Gly Leu Ala Trp Ser Phe Val Thr Tyr Tyr

195 200 205

 

 

Ser Pro Asn Asp Val Arg Val Val Ala Glu Gly Phe Asp Phe Ala Asn

210 215 220

 

 

Gly Ile Asn Ile Ser Pro Asp Gly Lys Tyr Val Tyr Ile Ala Glu Leu

225 230 235 240

 

 

Leu Ala His Lys Ile His Val Tyr Glu Lys His Ala Asn Trp Thr Leu

245 250 255

 

 

Thr Pro Leu Lys Ser Leu Asp Phe Asp Thr Leu Val Asp Asn Ile Ser

260 265 270

 

 

Val Asp Pro Val Thr Gly Asp Leu Trp Val Gly Cys His Pro Asn Gly

275 280 285

 

 

Met Arg Ile Phe Tyr Tyr Asp Pro Lys Asn Pro Pro Gly Ser Glu Val

290 295 300

 

 

Leu Arg Ile Gln Asp Ile Leu Ser Glu Glu Pro Lys Val Thr Val Val

305 310 315 320

 

 

Tyr Ala Glu Asn Gly Thr Val Leu Gln Gly Ser Thr Val Ala Ala Val

325 330 335

 

 

Tyr Lys Gly Lys Leu Leu Ile Gly Thr Val Phe His Lys Ala Leu Tyr

340 345 350

 

 

Cys Glu Leu

355

 

 

Claims (5)

1. an expression and purification method for PON1 gene, it is characterized in that, step is as follows:

(1) increase PON1 gene, is connected to transfer vector pFastBac ^tMrecombinant transfer vector is built on 1, recombinant transfer vector transformation of E. coli DH10Bac competent cell, obtain the recombinant baculovirus DNA containing PON1 gene, make it be assembled into the recombinant virus containing PON1 gene in time multiplexed cell system recombinant baculovirus DNA transfection Bombyx noriN cell;

(2) the recombinant virus inoculation silkworm five-age larva of step (1), gets silkworm blood after morbidity, purifies silkworm blood through centrifuging removal of impurities and after concentrated, acquisition PON1 albumen by ciba blue affinity chromatography and antibody affinity chromatography.

2. the expression and purification method of PON1 gene according to claim 1, is characterized in that, the primer nucleotide sequences that the described amplification of step (1) uses is as shown in SEQ ID NO:1 ~ 2.

3. the expression and purification method of PON1 gene according to claim 1, is characterized in that, step (2) described centrifugal be with centrifugal 30 min of the rotating speed of 10000 rpm under 4 DEG C of conditions; Described filtering and impurity removing is again through 0.45 μm of filter membrane after employing 9 layers of hospital gauze filter; Described concentrating is carried out with 10 kD super filter tubes.

4. the expression and purification method of PON1 gene according to claim 1, is characterized in that, the stationary phase that the described ciba blue affinity chromatography of step (2) uses is ciba blue agarose-3GA3000-CL.

5. the expression and purification method of PON1 gene according to claim 1, it is characterized in that, step (2) described antibody affinity chromatography antibody used gets blood after PON1 gene four immunize New Zealand male rabbits by prokaryotic expression then to be obtained by ProteinA purifying.