CN102925415A - Silkworm recombination baculovirus expressing paraoxonase 1 gene and preparation method and application thereof - Google Patents

Abstract

The invention relates to silkworm recombination baculovirus expressing a paraoxonase 1 gene and a preparation method and application thereof and belongs to the technical field of biopharmacy. By means of correlation technique of gene engineering, the paraoxonase 1 gene is built in a silkworm baculovirus expressing system, and then a silkworm bioreactor is used for expressing target protein. The silkworm bioreactor is used for expressing the paraoxonase 1 gene and conducting activity detection of the paraoxonase 1 gene, efficient expression of the protein is achieved, modification such as glycosylation is increased compared with the target protein expressed through prokaryotic expression, and foundation for further researching functions of the target protein is provided.

Description

Express silkworm with recombinant baculovirus of PON1 gene and its preparation method and application

Technical field

The present invention relates to a kind of silkworm with recombinant baculovirus of expressing the PON1 gene and its preparation method and application, belong to biological pharmacy technical field.

Background technology

PON1 (paraoxonase1, PON1) extensively is present in sialisterium behind multiple-microorganism, insect, snake venom, squid axon, the squid, birds and the Mammals, is the non-specific esterase of a kind of wide spectrum of existing in the organism.It can come degrading organic phosphor acid, aromatic hydroxy group acid esters and carbamate by the hydrolysis of catalysis phosphoric acid ester bond, thereby hydrolysis organic phosphorous insecticide and degraded nerve poison are played a significant role.Therefore PON1 is expected to become the new way of organophosphate poisoning prevention and treatment.It is reported, PON1 not only plays an important role in the organophosphorus toxicants detoxifcation, and with morbidity of mammiferous atherosclerosis, coronary heart disease and type II diabetes etc. substantial connection is arranged, the all right cancellation colony induction signaling molecule of while PONs, disturb bacterial population induced signal (the Quorum sensing of system, QS), this discovery has disclosed the new mechanism of Mammals bacterial-infection resisting.

Current terrorism is spread unchecked, the event of organophosphate poisoning and suicide happens occasionally; to adopt coromegine and Pralidoxime Chloride for the traditional methods for the treatment of of neurotoxic; Pseudocholinesterase by indirect inhibition m receptor and resurrection phosphorylated reaches the detoxifcation purpose, but has the deficiencies such as atropinism and the knock-on of poisoning in the treatment.Because PON1 can the multiple organophosphorus compound of direct hydrolysis, and its hydrolysate can directly go out from internal metabolism, so the discovery of PON1 very likely provides a new approach for prevention and the treatment of organophosphate poisoning.

Recent study finds, PON1 also has the effect that suppresses LDL oxidation, with atherosclerosis, chronic hepatic injury, coronary heart disease and type II diabetes etc. the morbidity of various diseases substantial connection is arranged.

Utilize the PON1 of escherichia coli expression that the advantage of himself is arranged, such as cheapness, with short production cycle etc., but the albumen of expressing lacks glycosylation and phosphorylation modification, have immunological rejection when using in vivo, the transformation period is short, and (escherichia coli prokaryotic expression system is not translated the post-treatment system to the deficiencies such as easy degraded, can not carry out the modifications such as glycosylation, phosphorylation, thereby make part can not in this system, carry out functional expression to the higher gene in glycosylation requirement Chengdu).(the Teng Xia such as Sun Manji, Yang Shen, before Lee, Sun Manji, Deng. Q-Type Human Paraoxonase Expression in Escherichia coli [J]. the biotechnology communication, 2007 (18): 15-18) once used intestinal bacteria system table intelligent Q Serum paraoxonase, the result shows and expressed albumen is all existed with the inclusion body form, after the step process such as the separation of inclusion body process, sex change, renaturation, product does not show enzymic activity.(the Ji Aiguo such as Ji Aiguo, the Asia, Huaihe River is red, Zhang Lupei, Deng. the structure of ox PON1 gene and paraoxonase 2 and in prokaryotic expression carrier is expression analysis [J] extremely. the biotechnology communication, 2008,35 (9): 59-63) clone the bovine serum paraoxonase gene, and utilize gene engineering method tentatively to give expression to the activated paraoxonase PON1 of tool, but do not obtain the recombinant protein of purifying.

Summary of the invention

In view of this, the object of the present invention is to provide a kind of silkworm with recombinant baculovirus of expressing the PON1 gene, in the silkworm bio-reactor, carry out high efficient expression by baculovirus expression system with PON1 is gene constructed, increased glycosylated function than prokaryotic expression, the natural modifications function of more expressing near Mammals, and silkworm can reduce the cost of exogenous gene expression greatly as a kind of cheap low-cost bio reactor.

For achieving the above object, the invention provides a kind of silkworm with recombinant baculovirus of expressing the PON1 gene, this virus contains the PON1 gene.

Preferably, the nucleotide sequence of wherein said PON1 gene is shown in SEQ ID NO:1.

The present invention also provides a kind of preparation method of above-mentioned silkworm with recombinant baculovirus, may further comprise the steps:

(1) the PON1 gene is synthetic;

(2) PON1 (PON1) gene that step (1) is synthesized is connected on the transfer vector, and transforms the e. coli tg1 competent cell, makes up recombinant transfer vector;

(3) recombinant transfer vector that step (2) is made up transforms intestinal bacteria DH10Bac competent cell, obtains recombinant baculovirus DNA;

(4) the recombinant baculovirus DNA transfection bombyx mori cell that step (3) is obtained obtains silkworm with recombinant baculovirus.

Preferably, wherein the synthetic of the described PON1 gene of step (1) specifically comprises:

(1) design upstream primer F:5 '-TAT GAATTCATGGCTAAACTGACAGCG-3 ', wherein italic is EcoR I site;

Downstream primer R:5 '-GCG CTCGAGTTACAGCTCACAGTAAAG-3 ', wherein italic is XhoThe I site;

(2) take the pBlueScript-PON1 plasmid as template, with designed primers F and R specific amplification fragment, specific procedure is: 95 ℃ of denaturation 5min, and 95 ℃ of sex change 45s, 55 ℃ of annealing 30s, 72 ℃ are extended 45s, circulate 30 times; Last 72 ℃ are extended 45s, reclaim product, obtain PON1 (PON1) gene, find that after the order-checking comparison gene order that obtains is consistent with design.

Preferably, wherein said step (2) specifically comprises:

PON1 (PON1) gene that pcr amplification obtains is used EcoRI and XhoReclaim behind the I double digestion, be connected to the transfer vector pFastBac through same processing ^TMOn the HTA, the PON1 gene is positioned under the control of polyhedrosis gene promotor, then is transformed in the e. coli tg1 competent cell, screening positive clone obtains recombinant transfer vector pFastBac ^TMHTA-PON1.

Preferably, wherein said step (3) specifically comprises:

The recombinant transfer vector pFastBacTMHTA-PON1 that step (2) is obtained transforms intestinal bacteria DH10Bac competent cell, obtain recombinant baculovirus DNA (Bacmid-PON1) by blue hickie screening, and utilize M13 upstream and downstream primer to carry out the PCR combination and identify.

Preferably, wherein the method for the described recombinant baculovirus DNA of step (4) transfection bombyx mori cell is: recombinant baculovirus DNA is changed in the bombyx mori cell by liposome mediated-method.

The albumen that the present invention also provides a kind of above-mentioned silkworm with recombinant baculovirus to express, its aminoacid sequence is shown in SEQ ID NO:2.

The present invention also provides the preparation method of above-mentioned albumen, may further comprise the steps:

(1) above-mentioned infecting silkworm with recombinant baculovirus Bombyx noriN cell is carried out virus amplification;

(2) needle punching inoculation silkworm five-age larva and silkworm chrysalis;

The above-mentioned albumen of (3) expressing wait the rear collection of falling ill.

The present invention further provides a kind of medicine, its activeconstituents is the albumen of aforesaid method preparation.

The present invention adopts baculovirus expression vector system to realize the expression of PON1, and its advantage mainly is:

1) expression amount is high: the high efficient expression that utilizes the strong promoter driving foreign gene of baculovirus polh gene and p10 gene; 2) have and transcribe, translate the post-treatment ability: baculovirus expression vector system has the very strong post-treatment ability of transcribing, translate, and expression product has very strong similarity at structure, biological activity, immunogenicity and the aspect such as glycosylation modified and native protein; 3) have benefited from the development of artificial incubation and artificial diet technology, silkworm can carry out extensive aseptic artificial breeding the whole year, produces in a large number useful matter; 4) security is good: insect baculovirus only copies in invertebral zooblast, be not pathogenic agent for vertebrates and vegetable cell, therefore safe and harmless (expressive host of silkworm baculovirus has bombyx mori cell, silkworm larva and silkworm chrysalis to insect baculovirus expression system to the user, but silkworm larva and silkworm chrysalis use range are wider, because it is convenient to injection operation); 4) utilize baculovirus expression vector system to express PON1, be conducive to solve the not enough problem in natural PON1 source, and it is active as the paraoxonase that eukaryotic expression system produces to study on this basis silkworm, can lay the foundation for further utilizing the neural toxinicide of silkworm biological reactor low cost production organophosphorus.

Description of drawings

Fig. 1 is the pcr amplification product electrophoretic analysis figure of PON1 gene; M:DNA ladder marker; 1: negative control; The 2:PCR product;

Fig. 2 is recombinant transfer vector pFastBac ^TMThe double digestion of HTA-PON1-PCR identifies figure M:DNA ladder marker; 1: the double digestion product; The 2:PCR product;

Fig. 3 identifies figure for the PCR of restructuring Bacmid-PON1; M:DNA ladder marker; The PCR product of 1:M13F/M13R; The PCR product of 2:M13F/PON1 downstream primer; The PCR product of 3:PON1 upstream primer/M13R; The PCR product of 4:PON1 upstream primer/PON1 downstream product;

Fig. 4 is the PCR evaluation figure of recombinant baculovirus BmNPV-PON1; M:DNA ladder marker; The PCR product of 1:M13F/M13R; The PCR product of 2:M13F/PON1 downstream primer; The PCR product of 3:PON1 upstream primer/M13R; The PCR product of 4:PON1 upstream primer/PON1 downstream product;

Fig. 5 is that recombinant baculovirus BmNPV-PON1 is at SDS-PAGE and the Western blotting evaluation figure of silkworm cells product; M: molecular weight of albumen standard; 1: the normal cell total protein; 2: the morbidity cell conditioned medium; 3: the morbidity cell precipitation;

Fig. 6 is SDS-PAGE and the Western blotting evaluation figure of recombinant baculovirus BmNPV-PON1 expression product in the silkworm five-age larva; M: molecular weight of albumen standard; 1: the silkworm hemolymph of inoculation wild virus morbidity; 2: the silkworm hemolymph supernatant of inoculation BmNPV-PON1 morbidity; 3: the silkworm hemolymph precipitation of inoculation BmNPV-PON1 morbidity;

Fig. 7 is SDS-PAGE and the Western blotting evaluation figure of recombinant baculovirus BmNPV-PON1 expression product in silkworm chrysalis; M: molecular weight of albumen standard; 1: normal silkworm chrysalis; 2: morbidity silkworm chrysalis supernatant; 3: morbidity silkworm chrysalis precipitation;

Fig. 8 is the active detection figure of PON1 that expresses in the bombyx mori cell;

Fig. 9 is the active detection figure of PON1 that expresses in the silkworm larva hemolymph;

Figure 10 is the active detection figure of the PON1 of expression in escherichia coli;

Figure 11 is the active detection figure of PON1 that exists in the rabbit anteserum.

Embodiment

Be noted that following specifying all is example, be intended to further specify the invention provides, except as otherwise noted, all Science and Technology terms used herein have the identical meanings of usually understanding with the technical field of the invention personnel.

The present invention will be further described below in conjunction with drawings and Examples.

Synthesizing of embodiment 1:PON1 gene

The PON1 gene order design pair of primers of announcing according to GenBank accession number AY499193.1 is used for amplification PON1 gene (italic is restriction enzyme site), and its nucleotide sequence is shown in SEQ ID NO:1.

Upstream primer F:5 '-TAT GAATTCATGGCTAAACTGACAGCG-3 ', wherein italic is EcoR I site;

Downstream primer R:5 '-GCG CTCGAGTTACAGCTCACAGTAAAG-3 ', wherein italic is XhoThe I site;

With pBlueScript-PON1 plasmid (Teng Xia, Yang Shen etc. Q-Type Human Paraoxonase Expression in Escherichia coli .[J] biotechnology communication 2007,18 (1): 015-018) be template, utilize above-mentioned primer to carry out pcr amplification, carry out agarose gel electrophoresis (as shown in Figure 1), the gene fragment size of PON1 is 1065bp approximately, and agarose gel electrophoresis shows that the PCR product size of No. 2 path goal gene is with actual consistent, reclaim product, obtain the PON1 gene.

Reaction system and response procedures arrange as follows:

Reaction system:

2×Mix 10μL；

Template 0.4 μ L;

Upstream primer 0.4 μ L;

Downstream primer 0.4 μ L;

ddH ₂O 8.8μL；

Total 20μL；

Response procedures:

Embodiment 2: recombinant transfer vector pFastBac ^TMThe structure of HTA-PON1

PON1 gene and pFastBac to embodiment 1 gained ^TMHTA carrier (Invitrogen) is used EcoRI and XhoI (Fermentas company) is carried out double digestion simultaneously, and agarose gel electrophoresis reclaims enzyme and cuts product; The PON1 gene fragment that reclaims is connected to the pFastBac that cuts through enzyme with Ligation high ligase enzyme (Fermentas company) ^TMOn the HTA carrier, goal gene (PON1 gene) is positioned under the control of polyhedron promotor, transforms e. coli tg1 competent cell (Fermentas company), screening positive clone obtains recombinant transfer vector pFastBac ^TMHTA-PON1.This recombinant transfer vector is carried out PCR and double digestion, product carries out agarose gel electrophoresis and identifies (as shown in Figure 2), No. 1 two bright bands appear in path double digestion product, and wherein one is the PON1 gene fragment of 1065bp, and another is fragment behind the plasmid enzyme restriction; Bright band in No. 2 paths is the PCR product of the PON1 gene fragment of 1065bp, and then the success of agarose gel electrophoresis preliminary evaluation plasmid construction sends to order-checking, knows through order-checking, and goal gene successfully is cloned into pFastBac ^TMOn the HTA carrier.

Embodiment 3: the structure of silkworm with recombinant baculovirus BmNPV-PON1

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

The recombinant transfer vector pFastBac that embodiment 2 is built ^TMHTA-PON1 is transformed in the intestinal bacteria DH10Bac competent cell (Fermentas company), obtain positive colony by blue hickie screening, utilize M13 universal primer and goal gene upstream and downstream primer to be combined into respectively performing PCR, the PCR product that takes a morsel carries out agarose gel electrophoresis (as shown in Figure 3), and electrophoresis result shows the Bacmid-PON1 positive colony that acquisition is correct.

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

A. in advance 8 ~ 12h Bombyx noriN cell that growth conditions is good is got 1ml with pipettor and is laid in the minicell culture dish (specification is 3.5cm);

B. get 10 μ L restructuring Bacmid-PON1 and 8 μ L liposomes in sterilized EP pipe, Sf-900 II SFM (1 *) serum free medium (being purchased from U.S. GIBCO company) that adds 100 μ L, incubated at room is 20min approximately, obtains the Bacmid-PON1/ liposome complex;

C. add 2mL Sf-900 II SFM (1 *) serum free medium (being purchased from U.S. GIBCO company) washed twice after adherent growth Bombyx noriN cell in good condition among the step a being discarded substratum, to remove foetal calf serum residual in the former substratum;

D. add fresh Sf-900 II SFM (1 *) serum free medium 1mL, then add Bacmid-PON1/ liposome complex 100 μ L, rock gently culture dish mixture is evenly distributed; Hatch in 28 ℃ of cell culture incubators about 6h and to add 10 μ L foetal calf serums (FBS) (be purchased from Austrian PAA company) in this substratum and continue to cultivate;

E.28 behind ℃ cultivation 3 ~ 5d, (typical disease symptom: cell becomes circle by spindle shape to the onset state of microscopically observation of cell, and kernel increases, and cell free is in substratum; And normal cell is arranged closely and is fusiformis, and nucleus is obvious, and the cell attachment growth obviously.The cell morbidity shows restructuring Bacmid success transfection Bombyx noriN cell, and successfully copies, assembles formation recombinant virus BmNPV-PON1 in cell).

Embodiment 4: the evaluation of silkworm with recombinant baculovirus BmNPV-PON1

1) PCR identifies

After the Bombyx noriN cell morbidity, collect the recombinant virus suspension, extract viral genome with UNIQ-10 pillar viral genome extraction agent box (giving birth to worker biotech firm available from Shanghai).Be combined into performing PCR with M13F/M13R, M13F/PON1 downstream primer, PON1 upstream primer/M13R, PON1 upstream primer/PON1 downstream primer respectively, the PCR product that takes a morsel carries out agarose gel electrophoresis (seeing Fig. 4), and electrophoresis result shows and obtained correct recombinant Bombyx mori baculovirus BmNPV-PON1.

2) SDS-PAGE and Western blotting Analysis and Identification

To identify the silkworm with recombinant baculovirus inoculation BmN cell that is positive through PCR, cultivate about 72h for 27 ℃, treat that (typical disease symptom: cell becomes circle by spindle shape, and kernel increases, and cell free is in substratum in cell morbidity (microscopic examination); And normal cell is arranged closely and is fusiformis, and nucleus is obvious, and the cell attachment growth obviously.The cell morbidity shows restructuring Bacmid success transfection Bombyx noriN cell, and successfully copies, assembles formation recombinant virus BmNPV-PON1 in cell).Rear collecting cell also carries out SDS-PAGE and Western blotting to cell expression product and analyzes, and the result specific band (as shown in Figure 5) occurs near being presented at 43kD, and this silkworm with recombinant baculovirus successful expression in Bombyx noriN cell is described.

Embodiment 5: SDS-PAGE and the Western blotting Analysis and Identification of silkworm with recombinant baculovirus BmNPV-PON1 inoculation silkworm five-age larva

Needle punching inoculation silkworm (mountain valley with clumps of trees and bamboo pine * bright moon) five-age larva is collected silkworm hemolymph frozen in-80 ℃ of refrigerators after the silkworm with recombinant baculovirus BmNPV-PON1 amplification after its morbidity.

The silkworm larva hemolymph treatment process of morbidity: after 9 layers of hospital gauze filtered, 4 ℃ through the centrifugal 10min of 2000rpm with hemolymph, collected supernatant, precipitated resuspended with 0.9% physiological saline.Silkworm hemolymph after processing is carried out SDS-PAGE and Western blotting analysis.The result shows, occurs specific band (as shown in Figure 6) in the supernatant near the 43kD, and silkworm with recombinant baculovirus successful expression in the silkworm five-age larva is described.

Embodiment 6: SDS-PAGE and the Western blotting Analysis and Identification of silkworm with recombinant baculovirus BmNPV-PON1 inoculation silkworm chrysalis

With inoculation silkworm chrysalis after the silkworm with recombinant baculovirus BmNPV amplification, frozen in-80 ℃ of refrigerators after the silkworm chrysalis morbidity.

The treatment process of morbidity silkworm chrysalis: take by weighing 200g morbidity silkworm chrysalis, add 200mL, 0.9% physiological saline carries out homogenate.Homogenate 2min, ice bath 2min, repetitive operation 10 times; Then behind 4 ℃ of centrifugal 30min of lower 4000rpm, remove degrease, repetitive operation 3 times with 9 layers of filtered through gauze.Silkworm chrysalis sample after processing is carried out SDS-PAGE and Western blotting analysis.The result shows, approximately specific band (as shown in Figure 7) occurs near the 43kD in the precipitation, and silkworm with recombinant baculovirus successful expression in silkworm chrysalis is described.

Embodiment 7: the activity of utilizing the natural PON1 that exists in that paraoxon detects bombyx mori cell, larva hemolymph, expression in escherichia coli as substrate and the rabbit anteserum

Can be hydrolyzed paraoxon according to paraoxonase and become p-nitrophenol (yellow compound, at the 405nm place absorption peak is arranged) principle, react the activity level of paraoxonase by the generating rate that detects p-nitrophenol, both at home and abroad the activity of paraoxonase being detected at present mainly is as substrate with paraoxon or phenylacetate.

Principle: take paraoxon (paraoxon) as substrate, 25 ℃ of incubations generate p-nitrophenol, detect absorbancy at 405nm wavelength place, generate p-nitrophenol 1 μ mol as a unit (U/L) take every liter of sample hydrolysis of per minute.

Method: adopt continuous monitoring method to measure PON1 active.

Concrete operations: in 96 orifice plates (Thermo Scientific company), respectively by following reaction system successively application of sample (annotate: testing sample should add reaction system at last), put into immediately the long microplate reader of all-wave (ThermoFisher Scientific Skan I+) that sets in advance response procedures behind the shaking table mixing and carry out the continuously measured of OD value.Response procedures: hatch 1min, 181 kinetics points of 25 ℃ of lower continuously measureds, the timed interval is 1min, the colorimetric command set is 405nm.Draw out the enzymatic reaction kinetics curve and carry out the calculating of enzyme activity according to formula according to the OD value of surveying.

The speed (△ A/min) that rises according to absorbancy calculates the result.The prescription of reaction system is shown in following table 1-4: (annotate: PON1 is calcium ion dependency esterase, and two Ca are arranged in the active centre of PON1 ²⁺, one of them participates in consisting of catalytic center, and is essential by keeping enzymic activity; Another plays an important role Ca aspect PON1 structure stable keeping ²⁺Can impel short-term formation enzyme-substrate complex and facilitate it to decompose, EDTA then can suppress the activity of PON1.)

Express the active (unit: μ L) that detects of PON1 in table 1 silkworm hemolymph

Express the active (unit: μ L) that detects of PON1 in table 2 bombyx mori cell

Active (the unit: μ L) that detects behind the PON1 purifying of table 3 escherichia coli expression

Active (the unit: μ L) that detects of natural PON1 in table 4 rabbit anteserum

(illustrate: table 3 is escherichia coli prokaryotic expression and purified PON1 determination of activity, only needs to suppress its activity with EDTA and gets final product as negative control; Table 4 is naturally occurring PON1 determinations of activity in the rabbit anteserum, and as positive control, purpose is that the PON1 activity with itself and silkworm expression compares, and EDTA only need to be set suppress its activity and get final product as negative control).

Calculation formula: PON1 (U/L)=△ A/min * Vt * 106/ (ε * Vs * L)

Wherein △ A/min is per minute absorbancy changing value; Vt is the reaction system cumulative volume; Vs is the testing sample volume, and L is cuvette optical path (1cm); 10 ⁶For mol being converted into the coefficient of μ mol; ε is the molar extinction coefficient 18750L/ (mol cm) of p-nitrophenol.

Calculation result: Fig. 8-11 is the enzymatic reaction kinetics curve, show be product OD value over time, choosing the numerical value that is linear change from Fig. 8-11 (is the larger part numerical value of speed of reaction, such as 1 ~ t min), deduct negative control generates the product p-nitrophenol as this time point OD value, (A _{T min}-A _1min)/(t min-1min) carries out ensuing calculating as the △ A/min in the formula.

Active detect (the seeing Fig. 8) of the PON1 that expresses in the bombyx mori cell:

Enchylema PON1 (U/L)=[0.003247/1min*200 μ l*10 ⁶]/(18750*20 μ l*1cm)=1.7317.

Active detect (the seeing Fig. 9) of the PON1 that expresses in the silkworm larva hemolymph:

Silkworm blood after the dilution

PON1(U/L)=[0.0026/1min*200μl*10 ⁶]/ (18750*20μl*1cm)=1.3867

Multiply by extension rate is 1.3867*2000 μ l/27 μ l=102.72.

Active detect (the seeing Figure 10) of the PON1 of expression in escherichia coli:

32a-PON1(U/L)=[0.099887/0.5min*200μl*10 ⁶]/(18750*10μl*1cm)=213.09。

Active detect (the seeing Figure 11) of naturally occurring PON1 in the rabbit anteserum:

Rabbit anteserum PON1 (U/L)=[0.6314/1min*200 μ l*10 ⁶]/(18750*20 μ l*1cm)=336.747.

Conclusion: by calculation result as can be known, though the PON1 detection of active of expression in escherichia coli up to 213.09U/L, its than eukaryotic expression albumen lack a series of protein modified functions such as glycosylation and phosphorylation, its biological activity remains further research; Bombyx mori cell is because without concentration, and concentration is lower, and naturally occurring PON1 activity is low in the specific activity rabbit anteserum that therefore detects, and the cell cultures cost is higher, therefore is not suitable for large scale culturing; And the PON1 activity of expressing in the process silkworm larva hemolymph of preliminary concentration is up to 102.72U/L, slightly lower than naturally occurring PON1 activity in the rabbit anteserum, and silkworm belongs to eukaryote, although protein modified degree may be slightly more inferior than Mammals, but it is cheap and easy to get, has very high researching value.

The above is embodiments of the invention only, should be understood that; for the those of ordinary skill in the present technique; under the prerequisite that does not break away from core technology feature of the present invention, can also do some improvements and modifications, these retouchings and improvement also should belong to scope of patent protection of the present invention.

SEQUENCE LISTING

＜110〉Tianjin Yaoyu Biotechnology Co., Ltd. Tianjin international bio medicine joint study institute

＜120〉silkworm with recombinant baculovirus of expression PON1 gene and its preparation method and application

<130> 122016-I-CP-TJYU

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 1065

<212> DNA

＜213〉PON1 gene

<400> 1

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> 2

<211> 355

<212> PRT

＜213〉PON1 albumen

<400> 2

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 (10)

1. a silkworm with recombinant baculovirus of expressing the PON1 gene is characterized in that, this virus contains the PON1 gene.

2. silkworm with recombinant baculovirus as claimed in claim 1 is characterized in that, the nucleotide sequence of described PON1 gene is shown in SEQ ID NO:1.

3. the preparation method of the described silkworm with recombinant baculovirus of claim 1 is characterized in that, the method may further comprise the steps:

(1) the PON1 gene is synthetic;

(2) the PON1 gene that step (1) is synthesized is connected on the transfer vector, and transforms the e. coli tg1 competent cell, makes up recombinant transfer vector;

(3) recombinant transfer vector that step (2) is made up transforms intestinal bacteria DH10Bac competent cell, obtains recombinant baculovirus DNA;

(4) the recombinant baculovirus DNA transfection bombyx mori cell that step (3) is obtained obtains silkworm with recombinant baculovirus.

4. method as claimed in claim 3 is characterized in that, the synthetic of the described PON1 gene of step (1) specifically comprises:

(1) design upstream primer F:5 '-TAT GAATTCATGGCTAAACTGACAGCG-3 ', wherein italic is EcoR I site;

Downstream primer R:5 '-GCG CTCGAGTTACAGCTCACAGTAAAG-3 ', wherein italic is XhoThe I site;

(2) take the pBlueScript-PON1 plasmid as template, with designed primers F and R specific amplification fragment, specific procedure is: 95 ℃ of denaturation 5min, and 95 ℃ of sex change 45s, 55 ℃ of annealing 30s, 72 ℃ are extended 45s, circulate 30 times; Last 72 ℃ are extended 45s, reclaim product, obtain PON1 gene PON1.

5. method as claimed in claim 3 is characterized in that, described step (2) specifically comprises:

The PON1 gene that pcr amplification obtains is used EcoRI and XhoReclaim behind the I double digestion, be connected to the transfer vector pFastBac through same processing ^TMOn the HTA, the PON1 gene is positioned under the control of polyhedrosis gene promotor, then is transformed in the e. coli tg1 competent cell, screening positive clone obtains recombinant transfer vector pFastBac ^TMHTA-PON1.

6. method as claimed in claim 3 is characterized in that, described step (3) specifically comprises:

The recombinant transfer vector pFastBac that step (2) is obtained ^TMHTA-PON1 transforms intestinal bacteria DH10Bac competent cell, obtains recombinant baculovirus DNA by blue hickie screening.

7. method as claimed in claim 3 is characterized in that, the method for the described recombinant baculovirus DNA of step (4) transfection bombyx mori cell is: recombinant baculovirus DNA is changed in the bombyx mori cell by liposome mediated-method.

8. the described silkworm with recombinant baculovirus of a claim 1 albumen of expressing, its aminoacid sequence is shown in SEQ ID NO:2.

9. the preparation method of the described albumen of claim 8 is characterized in that, the method may further comprise the steps:

(1) the arbitrary described infecting silkworm with recombinant baculovirus Bombyx noriN cell of claim 1-2 is carried out virus amplification;

(2) needle punching inoculation silkworm five-age larva and silkworm chrysalis;

The right 8 described albumen of (3) expressing wait the rear collection of falling ill.

10. a medicine is characterized in that, its activeconstituents is the albumen according to the described method preparation of claim 9.

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