CN102516369A

CN102516369A - Mutant polyhedron and preparation method thereof

Info

Publication number: CN102516369A
Application number: CN2011104320433A
Authority: CN
Inventors: 杜君卿; 陈剑清; 舒特俊; 张耀洲
Original assignee: TIANJIN YAOYU BIOLOGICAL TECHNOLOGY Co Ltd
Current assignee: TIANJIN YAOYU BIOLOGICAL TECHNOLOGY Co Ltd
Priority date: 2011-12-21
Filing date: 2011-12-21
Publication date: 2012-06-27

Abstract

The invention relates to a mutant polyhedron with which a target protein yield can be improved. The invention related to the field of biology. According to the invention, a recombinant expression vector pET-28a-polh is constructed; the recombinant expression vector is mutated by using a commercialized kit, such that a complete recombinant expression vector containing mutant sites is obtained; the complete recombinant expression vector is subject to induction expression, such that the mutant polyhedron which can reduce a dissolving pH is obtained. When the mutant polyhedron provided by the invention is connected to a target protein gene, an obtained fusion protein can be purified through pH regulation and differential centrifugation methods with the property of the polyhedron, such that the purification steps are simplified. The obtained fusion protein can also be digested under an optimal protease active pH condition, such that target protein with a maximal amount can be obtained. Therefore, the yield of target protein is greatly improved.

Description

A kind of sudden change polyhedrin and preparation method thereof

Technical field

The present invention relates to a kind of sudden change polyhedrin, relate in particular to a kind of can improve the target protein yield sudden change polyhedrin and preparation method thereof.

Background technology

Because polyhedrin has the ability that efficiently expresses, therefore often with it as a kind of label, will not express or difficult expressed proteins gene fusion gets on, thereby it is expressed or improve its expression amount.Simultaneously, can utilize polyhedrosis dissolution characteristics, the method through simple pH regulator and differential centrifugation obtains purer fusion rotein, carries out proteolytic cleavage afterwards and obtains pure target protein.But; The best use of pH scope of proteolytic enzyme is at 7.0-8.5, and polyhedrin only just can dissolve when pH10.8, so under this condition, carry out enzyme when cutting; The proteolytic enzyme loss of activity perhaps can't reach maximum activity, and the amount of resulting target protein also is restricted.

Summary of the invention

Cut the lower deficiency of efficient in order to overcome in the existing purification process proteolytic enzyme enzyme, the present invention provides a kind of sudden change polyhedrin that utilizes overlapping extension PCR method to obtain.It has reduced the dissolving pH of polyhedrin effectively, polyhedrin is cut under the pH condition at the best enzyme of proteolytic enzyme be in dissolved state; Utilizing polyhedrin as the label purified fusion protein, when through proteolytic enzyme enzyme blanking method polyhedron being removed at last, proteolytic enzyme just can be brought into play its maximum activity, thereby obtains a large amount of target proteins.

The present invention's technical scheme that is adopted of dealing with problems is: through the design primer; All albumen labels on the pET-28a carrier are removed; Merge the wild-type polyhedron gene of entering then, purpose is that to make the expression product that obtains be the polyhedrin that does not contain any label protein.Then; Design contains the primer in mutational site, utilizes commercial rite-directed mutagenesis test kit, and 4 successive basic aminoacidss that N in the wild-type polyhedrin aminoacid sequence is held sport acidic amino acid; To this be connected with the sudden change polyhedron gene expression vector carry out abduction delivering after; Method through traditional adjusting pH is carried out purifying, and with the polyhedrin of wild-type relatively, just be in solvable state when detecting polyhedrin through sudden change at pH8.5.

For achieving the above object, the present invention provides a kind of sudden change polyhedrin, and its aminoacid sequence is shown in SEQ ID NO:4.

The preparation method of said mutation polyhedrin may further comprise the steps:

(a) method by pcr amplification obtains the wild-type polyhedron gene;

(b) through the design primer, all the albumen labels on the pET-28a carrier are removed, and connected the upward resulting polyhedron gene of step (a), be built into recombinant expression vector pET-28a- Polh

(c) utilize commercial rite-directed mutagenesis test kit, 4 successive basic aminoacidss of N end in the wild-type polyhedrin aminoacid sequence are sported acidic amino acid, and design contains the primer in mutational site;

(d) obtain recombinant expression vector pET-28a- PolhAfter, utilize commercial rite-directed mutagenesis test kit, carry out pcr amplification through step (c) gained mutant primer, obtain the complete recombinant expression vector that contains the mutational site;

(e) change step (d) gained recombinant expression vector over to e. coli bl21 and carry out abduction delivering, obtain said sudden change polyhedrin.

Further, in the above-mentioned steps (c) 32-35 position 4 the successive basic aminoacidss KRKK in the wild-type polyhedrin aminoacid sequence (shown in SEQ ID NO:2) has been mutated into acidic amino acid ESEE.

Further, the sudden change polyhedrin that obtains in the above-mentioned steps (e) can dissolve when pH8.5, and the wild-type polyhedrin can dissolve when pH10.8.

The invention has the beneficial effects as follows; On the one hand can continue to utilize polyhedron to make as the albumen label does not express or difficult expressed proteins is able to express or improve its expression amount; When polyhedrin being excised, can make its maximum activity of proteolytic enzyme performance on the other hand, obtain a large amount of target proteins with proteolytic enzyme.

Description of drawings

Fig. 1 is pET-28a-of the present invention PolhPlasmid map;

Fig. 2 is pET-28a-of the present invention PolhOverlapping extension PCR figure; M:DNA Ladder Mix; The 1:PCR amplified band;

Fig. 3 is sudden change polyhedrin expression figure of the present invention; M: molecular weight of albumen standard; The 1:pET-28a zero load is not induced; The 2:pET-28a zero load is induced; 3:pET-28a- PolhDo not induce; 4:pET-28a- PolhInduce;

Fig. 4 is the pH regulator figure of sudden change polyhedrin of the present invention;

M: molecular weight of albumen standard; 1:pH8.5, the 4000rpm supernatant; 2:pH8.5, the supernatant after the 4000rpm supernatant uses 15000rpm centrifugal once more.

Embodiment

Be noted that following specifying all is exemplary, being intended to provides further invention to the present invention.Except as otherwise noted, all Science and Technology terms of using of this paper have with the present invention under the identical meanings of person skilled common sense.

Below in conjunction with accompanying drawing and embodiment the present invention is further specified.

Embodiment 1: design of primers

Amplification wild-type polyhedrosis gene, design of primers is following:

Upstream primer F:5'-CATG CCATGGCCAATTATTCATACACCC-3' (represent by italic NcoThe I restriction enzyme site)

Downstream primer R:5'-CG GGATCCATACGCCGGACCAGTGAA-3' (represent by italic BamH I restriction enzyme site).

The embodiment 2:PCR wild-type polyhedrosis gene (shown in SEQ ID NO:1) that increases

With Bombyx mori nuclear polyhydrosis virus genome (available from Invitrogen company), the upstream primer F of embodiment 1 gained, downstream primer R are primer, amplification purpose fragment.The PCR reaction parameter is designed to, 94 ℃ of preparatory sex change 5min, and 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ of renaturation are extended 1min, 30 circulations, 72 ℃ are extended 10min.

In the centrifuge tube of 100 μ L, add following component:

KOD Dash Buffer 5μL

2.5mMdNTPs 1.5μL

KOD Dash 1μL

F 1μL

R 1μL

Template 1 μ L

Add aseptic double-distilled water to 50 μ L.

Behind each component mixing, put into the PCR appearance, by 30 circulations of above-mentioned reaction parameter design.After question response finished, electrophoresis was identified amplified fragments, cut glue simultaneously and reclaimed the purpose fragment.

Embodiment 3: the design of mutant primer

According to foregoing principle, hold continuous 4 basic aminoacids KRKK to sport 4 successive acidic amino acid ESEE the N of wild-type polyhedrin (shown in SEQ ID NO:2), following with the Primer5.0 designed primer:

Upstream primer F ': 5 '-GTGCTCCTCGCTCTCGGCGTTTTTGATAAG-3 '

Downstream primer R ': 5 '- GAG AGC GAG GAGCACCTAGTCGAACATGA-3 ' (bolded section is the mutational site).

Embodiment 4: make up recombinant expression vector pET-28a- Polh

The PCR product warp that embodiment 2 obtains NcoI with BamGene fragment clone behind the H I double digestion is to warp NcoI with BamAmong the carrier pET-28a of H I double digestion (Invitrogen company), the gene of pcr amplification is connected on the carrier pET-28a, is built into recombinant expression vector pET-28a- Polh(see figure 1), entirely true through restriction analysis identified gene sequence.

Embodiment 5: recombinant expression vector pET-28a- PolhSudden change

Obtain recombinant expression vector pET-28a- PolhAfter; Utilize commercial rite-directed mutagenesis test kit (Transgene company), carry out pcr amplification, obtain the complete expression vector that contains the mutational site and (wherein contain sudden change polyhedron gene fragment through embodiment 3 gained mutant primers; Shown in SEQ ID NO:3), the result is as shown in Figure 2.

Embodiment 6: the expression of sudden change polyhedrin (shown in SEQ ID NO:4)

The recombinant expression vector that contains the mutational site through double digestion identify correct after, again through order-checking, proving that the mutant nucleotide sequence of its sequence and design is identical promptly suddenlys change successfully.Subsequently, change recombinant expression vector over to e. coli bl21 (Invitrogen company) and carry out abduction delivering, collect thalline behind the 4h; The centrifugal 10min of 12000rpm, collecting precipitation, the 1 * PBS that adds 50 μ L is resuspended; 2 * the sample-loading buffer that adds 50 μ L then carries out specimen preparation, get 15 μ L and walk 12% SDS-PAGE protein electrophoresis, and with the expression vector of sky as contrast; The result is as shown in Figure 3, and sudden change polyhedrin (about 29kDa) obtains great expression.

Embodiment 7: the purifying of sudden change polyhedrin

Obtain suddenling change behind the polyhedrin; Utilize the method for traditional adjusting pH to carry out purifying; And contrast with the polyhedron of wild-type, the result is as shown in Figure 4, shows that the polyhedrin solvability of sudden change changes; In the supernatant of pH8.5, exist very in a large number, this is that the wild-type polyhedron is not available.

After sudden change polyhedron gene of the present invention connects the target protein gene; The fusion rotein that obtains can utilize polyhedrosis character to simplify purification step through the method for pH regulator and differential centrifugation; Can under proteolytic enzyme the best use of pH condition, carry out enzyme again cuts; Obtain the target protein of maximum, greatly improved the yield of target protein.

The above is merely the preferred embodiments of the present invention, 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 characteristic of the present invention, can also make some improvement and retouching, these retouchings and improvement also should belong to scope of patent protection of the present invention.

SEQUENCE LISTING

< 110>Tianjin Yaoyu Biotechnology Co., Ltd.

< 120>a kind of sudden change polyhedrin and preparation method thereof

<130> 111706-I-CP-TJYU

<160> 4

<170> PatentIn version 3.5

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gataccatga agttaatcgt caactggagc ggcaaagagt ttttgcgtga aacttggacc 300

cgttttgttg aggacagctt ccccattgta aacgaccaag aggtgatgga cgtgtacctc 360

gtcgccaacc tcaaacccac acgccccaac aggtgctaca agttcctcgc tcaacacgct 420

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actggtccgg cgtattaa 738

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Met Pro Asn Tyr Ser Tyr Thr Pro Thr Ile Gly Arg Thr Tyr Val Tyr

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Asp Asn Lys Tyr Tyr Lys Asn Leu Gly Cys Leu Ile Lys Asn Ala Lys

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Arg Lys Lys His Leu Val Glu His Glu Gln Glu Glu Lys Gln Trp Asp

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Leu Leu Asp Asn Tyr Met Val Ala Glu Asp Pro Phe Leu Gly Pro Gly

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Lys Asn Gln Lys Leu Thr Leu Phe Lys Glu Ile Arg Ser Val Lys Pro

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Asp Thr Met Lys Leu Ile Val Asn Trp Ser Gly Lys Glu Phe Leu Arg

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Glu Thr Trp Thr Arg Phe Val Glu Asp Ser Phe Pro Ile Val Asn Asp

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Gln Glu Val Met Asp Val Tyr Leu Val Ala Asn Leu Lys Pro Thr Arg

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Pro Asn Arg Cys Tyr Lys Phe Leu Ala Gln His Ala Leu Arg Trp Glu

130 135 140

Glu Asp Tyr Val Pro His Glu Val Ile Arg Ile Val Glu Pro Ser Tyr

145 150 155 160

Val Gly Met Asn Asn Glu Tyr Arg Ile Ser Leu Ala Lys Lys Gly Gly

165 170 175

Gly Cys Pro Ile Met Asn Ile His Ser Glu Tyr Thr Asn Ser Phe Glu

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Ser Phe Val Asn Arg Val Ile Trp Glu Asn Phe Tyr Lys Pro Ile Val

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Tyr Ile Gly Thr Asp Ser Ala Glu Glu Glu Glu Ile Leu Ile Glu Val

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Ser Leu Val Phe Lys Ile Lys Glu Phe Ala Pro Asp Ala Pro Leu Phe

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Thr Gly Pro Ala Tyr

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