CN102827858A

CN102827858A - Quick clone containing GB1 protein fusing label and expression plasmid vector

Info

Publication number: CN102827858A
Application number: CN2012103366896A
Authority: CN
Inventors: 邹培建; 盖园明; 赵普亚; 秦刚
Original assignee: Tianjin Institute of Industrial Biotechnology of CAS
Current assignee: Tianjin Institute of Industrial Biotechnology of CAS
Priority date: 2012-09-12
Filing date: 2012-09-12
Publication date: 2012-12-19

Abstract

The invention relates to a clone and a protein expression plasmid vector and comprises a section of green fluorescent protein (GFP) gene segment containing TEV enzyme recognition sites, being used for coding a gene sequence of a GB1 protein and being provided with BsaI enzyme cutting sites on two sides. The carrier constructed by the clone can achieve quick and high-efficient cloning and protein expression of genes under a condition that ligase is depended on. The GB1 fusing protein labels and TEV enzyme cutting sites are introduced in the carrier, the GB1 fusing protein labels help to increase solubility of protein which is difficult to dissolve and is favorable for separation and purification of the protein. A TEV enzyme recognition sequence is located on a downstream of the gene sequence of the GB1 protein, purified protein is cut through the TEV enzyme, and the GB1 fusing protein labels can be cut off to obtain a natural target protein. The TEV enzyme is an economic and practical enzyme at present so that test cost is greatly saved.

Description

A kind of quick clone and expression plasmid carrier that contains GB1 albumen fusion tag

Technical field

The invention belongs to technical field of molecular biology, be specifically related to a kind of clone and protein expression plasmid carrier, promptly a kind of quick clone and protein expression plasmid carrier that contains GB1 albumen fusion tag.

Background technology

Traditional cloning process is through plasmid vector is produced the sticky end of 2-4 base respectively with identical double digestion digestion back with target gene fragment; Utilize ligase enzyme that the complementary sticky end is connected; The subsequent transformation Bacillus coli cells, expression and purification albumen.Because sticky end length is shorter, follow-up must carrier the connection with target gene fragment through the T4 dna ligase, and in this process, possibly have the generation of part carrier from connecting.This method clone gene approximately needs two to three day time.In order to realize not relying on the cloning process of ligase enzyme, just need to make up a kind of convenient effective cloning vector.And the fusion tag that commercialization carrier in the market can utilize is also very limited, also all compares target protein expensive with the isolating protease price of label with what design on the carrier.Based on the consideration of above-mentioned several respects, be necessary to provide a kind of quick clone and protein expression vector that has fusion rotein label commonly used.

Summary of the invention

The object of the present invention is to provide a kind of clone and protein expression plasmid carrier, promptly a kind of the end at N contained GB1 fusion rotein label and can the gene of target protein be carried out the carrier of quick clone and protein expression, thereby remedies the deficiency of prior art.

Described plasmid vector of the present invention includes:

One section contains the TEV enzyme recognition site, and be used to encode the proteic gene fragment of GB1;

Both sides have the GFP gene fragment of BsaI restriction enzyme site.

The proteic nucleotides sequence of above-mentioned GB1 is classified SEQ ID NO:1 as.

Above-mentioned both sides have the GFP gene fragment of BsaI restriction enzyme site, and its nucleotides sequence is classified SEQ ID NO:2 as.

Carrier of the present invention also includes transcripting promoter, transcription terminator, kantlex encoding sequence and replicon fragment.

Carrier of the present invention, its genetic map is as shown in Figure 1, and nucleotides sequence is classified SEQ ID NO:3 as.

The plasmid vector that the present invention makes up is used at former pyrenomycetes express recombinant protein.

The carrier that the present invention makes up can not rely on clone rapidly and efficiently and the protein expression of realizing goal gene under the situation of ligase enzyme.And in carrier, introduced GB1 fusion rotein label and TEV restriction enzyme site, GB1 fusion rotein label helps to increase the protelytic solubleness of indissoluble, is beneficial to proteic separation and purification; The TEV enzyme recognition sequence is positioned at the downstream of the proteic gene order of GB1, through the TEV enzyme purified proteins matter is cut, and can the excision of GB1 albumen label be obtained natural target protein.The TEV enzyme is present most economical suitable enzyme, thereby has greatly practiced thrift experimental cost.

Description of drawings

Fig. 1: the genetic map of plasmid vector pJM-GB1 of the present invention;

Fig. 2: the expression and purification protein graphical spectrum of recombinant protein pJM-GB1;

Wherein: M, PageRuler ^TMPrestained Protein Ladder; 1, induces the total protein of preceding BL21/pJM-GB1; 2, induce after ultrasonic degradation and centrifugal after the supernatant part; 3, induce after ultrasonic degradation and centrifugal after the deposition part; 4, affinity chromatography stream is worn liquid; 5,20mM imidazoles washings; 6,200mM imidazoles elutriant.

Embodiment

Carrier of the present invention is the plasmid that sets out with pET-24d, in building process, has added the GB1 protein gene respectively, TEV restriction enzyme site and GFP gene.Respectively introduced a BsaI single endonuclease digestion site in GFP gene both sides.On the one hand; Through the BsaI single endonuclease digestion; This carrier respectively produces the sticky end of 4 bases that 5 ' distal process goes out by linearizing and in linearizing fragment both sides, after handling through T4 DNA Polymerase and dTTP, the base number of sticky end respectively increases to 10 and 12; Utilize this sticky end, can make things convenient for quick will goal gene be cloned on this carrier and carry out proteic expression and purification.On the other hand, for faster, quality is cloned in control more easily; The GFP gene that in the middle of clone's restriction enzyme site BsaI site, inserts; In the process of follow-up screening positive clone, can judge whether clone gene is inserted in the middle of the carrier through observing whether produce green fluorescence.

Facing vector construction process of the present invention and application down is described in detail.

One, the structure of expression plasmid carrier (pJM-GB1)

The first step adds his-Tag-GB1 label and TEV restriction enzyme site at the N end of the MCS of pET-24d (buy from Novogen company, numbering 69772-3).At first complete sequence synthesizes his-tag-GB1-TEV; Add the restriction enzyme site of XbaI and NcoI when synthetic at the two ends of his-tag-GB1-TEV nucleotide sequence respectively, be connected to (wherein the his-tag-GB1-Tag-TEV sequence is SEQ ID NO:1) on the cloning vector.The cloned plasmids of preparation obtains including the small segment of his-Tag-GB1 label after with XbaI and NcoI double digestion; Handling the carrier that obtain through XbaI with the NcoI double digestion with pET-24d is connected; Obtain the plasmid that the N end has his-Tag-GB1 label and TEV restriction enzyme site through screening, temporary transient called after pET-24d-GB1.

In second step, in the middle of two above-mentioned plasmid pET-24d-GB1 that build of BsaI restriction enzyme sites introducing, replace MCS wherein.

Design primer primer requires 5 ' end through phosphorylation modification when synthetic

Upstream primer: 5 '-GGTCTCACCGCGTCGGGTCACCACCACCACCAC-3 '

Downstream primer: 5 '-CGGTCTCATGGCGCCCTGAAAATAAAGATTCTC-3 '

With pET-24d-GB1-Tag is that template increases, and the PCR product utilizes the T4 ligase enzyme to connect after reclaiming purifying, and screening obtains having the plasmid pET-24d-GB1-2BsaI of BsaI restriction enzyme site.

In the 3rd step, according to the gene order of GFP albumen (SEQ ID NO:2), the nucleotide sequence of synthetic BsaI-GFP-BsaI is to cloning vector.The cloning vector of preparation is cut the fragment that comprises GFP that obtains after the processing with the BsaI enzyme, passes through the carrier that the BsaI enzyme cuts after the processing with pET-24d-GB1-2BsaI and is connected, and obtains final plasmid pJM-GB1.

The genetic map of the plasmid pJM-GB1 of preparation is as shown in Figure 1, and its nucleotides sequence is classified SEQ ID NO:3 as.

Two, the effect of plasmid of the present invention detects

Plasmid vector provided by the invention respectively comprises the restriction enzyme site (black matrix is represented) of a BsaI in its GFP gene outside; This carrier is after the BsaI single endonuclease digestion is removed the GFP gene; Carrier is by linearizing, and respectively produces the sticky end of 4 bases that 5 ' distal process goes out in linearizing fragment both sides.Carrier after the BsaI enzyme is cut is handled through T4 DNA polymerse and dTTP; Utilize 3 ' → 5 ' 5 prime excision enzyme activity of T4 DNA polymerse; Can remove 3 ' end base and stop up to running into T, plasmid vector is increased to 10 and 12 by the sticky end base number at linearizing and two ends thus.Specific annealing can take place with complementary strand not relying under the situation of ligase enzyme in this sticky end.

Concrete experimental procedure:

1.BsaI digested plasmid carrier

The configuration mixed system:

5 μ g plasmid vectors

2.5μl?BsaI(10units/μl)

5μl?10×NEB?buffer?3

The sterilized water polishing is to 50 μ l

50 ℃ of enzymes are cut 1h.

0.8% agarose electrophoresis detects enzyme and cuts effect, and glue reclaims the plasmid vector fragment.

2.T4 DNA polymerse handles plasmid vector

The configuration mixed system:

600ng BsaI digestion back carrier

0.5μl?dTTP(100mM)

1μl?DTT(100mM)

0.2μl?100×BSA

0.4μl?T4?DNA?polymerse(3units/μl)

2μl?10×NEB?buffer?2

The sterilized water polishing is to 20 μ l

Hatch 30min for 22 ℃.Hatch 20min for 75 ℃, deactivation T4 DNA polymerse.

The preparation target gene fragment

2.2.1 the primer of purpose of design gene

Upstream primer: CAGGGCGCC ATG-goal gene

Downstream primer: GACCCGACGCGG TTA-goal gene

The upstream and downstream primer contains with the terminal complementary base sequence of plasmid carrier viscosity, contains the ATG initiator codon in the upstream primer, and downstream primer contains the TAA terminator codon.

2.2.2 utilize T4 DNA polymerse to handle the PCR product

The primer that utilization designs; With the goal gene is that template is carried out pcr amplification, after the product that amplification is come out reclaims, utilizes T4 DNA polymerse and dATP to handle; Utilize 3 ' → 5 ' 5 prime excision enzyme activity of T4 DNA polymerse, can remove 3 ' end base and stop up to running into A.

2.3 target gene fragment connects with carrier fast

Get the 1.5ml centrifuge tube, operate according to following method:

Get the plasmid vector (25-50ng) that 1 μ l handles well, target gene fragment (0.02pmol) mixing that same 2 μ l handle well is placed on 22 ℃ and hatches 5min.Add 1 μ l EDTA (25mM), mixing is placed on 22 ℃ and continues to hatch 5min.

Utilize the new clone of plasmid construction of the present invention, finish only to need 1h to accomplish to the completion that connects from PCR.And traditional method need generally need the 1d time through target gene fragment being carried out utilize the T4 dna ligase to connect again behind the double digestion.

2.4 transform

To connect product Transformed E .coli DH5a competent cell.

2.5 evaluation positive colony

Identify positive colony through colony polymerase chain reaction (PCR) method, order-checking is indicated as the purpose positive colony.

Plasmid vector transformed host cells of the present invention is a Bacillus coli cells, and best Bacillus coli cells is BL21, BL21 (DE3) etc.Expression plasmid pJM-GB1 is transformed into e. coli bl21, is cultured to OD600 with the LB substratum that contains 50 μ g/ml kantlex at 37 ℃ and is about 0.6, adds final concentration and induces for 0.3mM IPTG, and expression product runs the SDS-PAGE electrophoresis.As shown in Figure 2, second swimming lane is the solubility expression amount of target protein, and the 6th swimming lane is the electrophoresis result behind the purifying; The above results shows the solubility expression amount of target protein and the needs that purity all satisfies experiment.

The successful deletant Protein S ortaseA (61-621) that gives expression to a kind of protein ligase enzyme (SortaseA) of plasmid that utilizes the present invention to make up, the solubility effect is fine.The primer of purpose of design gene at first: upstream primer 5 '-CAGGGCGCCAT GGCATATTTGT-3 ' downstream primer 5 '-GACCCGACGCGGTTATT TGACTTCT-3 '; With the plasmid that contains the Sortase gene is that template is carried out PCR; Utilize BsaI to carry out enzyme after the PCR product reclaims and cut processing; Be connected with the pJM-GB1 carrier, Transformed E .coli DH5a, screening obtains positive colony.The plasmid Transformed E .coli BL21 that order-checking is correct, the IPTG inducible protein is expressed, and utilizes nickel post affinity chromatography that this albumen is carried out purifying, through the TEV enzyme purified proteins matter is cut, and with the excision of GB1 albumen label, obtains natural target protein.The TEV enzyme is present most economical suitable enzyme, and this has also greatly practiced thrift experimental cost.

Claims

1. clone and protein expression plasmid carrier for one kind, it is characterized in that described plasmid vector includes:

1) one section contains the TEV enzyme recognition site, and be used to encode the proteic gene fragment of GB1;

2) both sides have the GFP gene fragment of BsaI restriction enzyme site.

2. carrier as claimed in claim 1, its characteristic are that more the nucleotides sequence of the proteic gene of described coding GB1 classifies SEQ ID NO:1 as.

3. carrier as claimed in claim 1, its characteristic are that more described both sides have the GFP gene fragment of BsaI restriction enzyme site, and its nucleotides sequence is classified SEQ ID NO:2 as.

4. carrier as claimed in claim 1, its characteristic are that more described plasmid vector also includes transcripting promoter, transcription terminator, kantlex encoding sequence and replicon fragment.

5. the described carrier of claim 1, its genetic map is as shown in Figure 1.

6. the described carrier of claim 1, its nucleotides sequence is classified SEQ ID NO:3 as.

7. the described carrier of claim 1 is used at former pyrenomycetes highly effective expressing recombinant protein.