CN103849638A

CN103849638A - Plasmid vector for constructing and screening cDNA (complementary deoxyribonucleic acid) library and construction method and application of plasmid vector

Info

Publication number: CN103849638A
Application number: CN201410058679.XA
Authority: CN
Inventors: 崔晓艳; 陈新; 智海剑; 顾和平; 袁星星; 张红梅; 陈华涛
Original assignee: Jiangsu Yanjiang Agricultural Science Research Institute
Current assignee: Jiangsu Yanjiang Agricultural Science Research Institute
Priority date: 2014-02-21
Filing date: 2014-02-21
Publication date: 2014-06-11

Abstract

The invention provides a plasmid vector for constructing and screening a cDNA (complementary deoxyribonucleic acid) library and a construction method and application of the plasmid vector. The most effective method for the interaction of a membrane protein and a host is the utilization of a ubiquitin-mediated membrane protein yeast two-hybrid system, and a convenient and rapid method for the construction of the cDNA library is a Gateway-based efficient library construction system. The membrane protein yeast two-hybrid system is fused with the Gateway system, a vector pPR3-N is used as a template, and an attR1-Cmr-ccdB-attR2 gene is introduced in an enzyme digestion connection way to transform a pPR3-gateway vector plasmid capable of efficiently constructing the cDNA library by using a Gateway technology, and the pPR3-gateway vector plasmid is used for the directional cloning, library construction and sequencing of full-length cDNA, or is used for the library screening of yeast two-hybrid.

Description

Build plasmid vector and construction process and the application of screening for cDNA library

Technical field

The invention belongs to gene engineering technology field, relate in particular to a kind of plasmid vector and construction process and application that builds screening for cDNA library.

Background technology

Proteomics research has been widely used in multiple systematic researches of doing mutually, comprise that virus studies with the mutual of host cell, and research method is also more and more diversified, common method mainly contains yeast two-hybrid, total length infectious clone, tandem affinity purification and bimolecular fluorescence complementary etc.For the membranin of encoding viral or the albumen of film correlation properties, owing to thering is film binding characteristic, mutual mostly occurring on film in vivo, traditional yeast two-hybrid system occurs in core, although added nuclear localization signal peptide on the carrier of test kit, gene all can be anchored in yeast core, but with cell in the situation of doing mutually under state of nature or discrepant, and posttranslational modification process etc. all cannot complete.Embrane-associated protein accounts for 1/3 in total cellular protein, and they have mostly participated in many physiology, pathologic process and the disease resistance response mechanism of cell.The interaction of research membranin has important meaning for disclosing the vital movement rule of cell and finding action target.For study the interactional needs of membranin in viable cell, successively develop the new system of a series of yeast two-hybrids for membranin repercussion study in the world in recent years, and obtained many important discoveries at medical field.There is at present the ubiquitin yeast two-hybrid system that is specially adapted for membranin, directly in film system, occurred to do mutually, solved these difficult problems.But membranin yeast two-hybrid system must be bought corresponding library or customization, cost is higher, and program complexity.

Summary of the invention

The object of the present invention is to provide a kind of plasmid vector that builds screening for cDNA library, be intended to Gateway technique to high-efficiency construction cDNA library plasmid, can transformed yeast, for membranin yeast two-hybrid.

A further object of the present invention is to provide the above-mentioned construction process that builds the plasmid vector of screening for cDNA library.

Another object of the present invention is to provide the above-mentioned application that builds the plasmid vector of screening for cDNA library.

The present invention is achieved in that a kind of plasmid vector that can be used for construction cDNA library screening, by pPR3-N carrier and attR1-Cm ^r-ccdB-attR2 genomic constitution, is designated as pPR3-gateway carrier, wherein, described attR1 sequence as shown in SEQ ID NO.1, described Cm ^rsequence is as shown in SEQ ID NO.2, and described ccdB sequence is as shown in SEQ ID NO.3, and described attR2 sequence is as shown in SEQ ID NO.4.

Preferably, described attR1-Cm ^r5 ' end of-ccdB-attR2 gene is connected with pPR3-N carrier through SfiI digestion with restriction enzyme respectively with 3 ' end.

Preferably, described attR1-Cm ^r-ccdB-attR2 gene is the sequence between the upper attR1 of carrier pDEST22 and attR2.

The construction process that the present invention further provides a kind of plasmid vector that can be used for construction cDNA library screening, comprises the following steps:

(1) for primers attR1sfiA and attR2sfiB between the upper attR1 of carrier pDEST22 and attR2, choose pPR3-N carrier universal sequencing primer thing pPR3-NF and pPR3-NR; Wherein, primer attR1sfiA and attR2sfiB sequence are as follows:

attR1sfiA:

5’-agtggccattacggccACAAGTTTGTACAAAAAAGCTGAACGAGAAACG-3’，

attR2sfiB:

5’-agaggccgaggcggccACCACTTTGTACAAGAAAGCTGAACGA-3’；

PPR3-NF and pPR3-NR primer sequence are as follows:

pPR3-NF：5’GTCGAAAATTCAAGACAAGG3’，

pPR3-NR：5’AAGCGTGACATAACTAATTAC3’；

(2) extension increasing sequence: take the DNA sequence dna of carrier pDEST22 as template, increase and obtain attR1-Cm with primer attR1sfiA and attR2sfiB ^r-ccdB-attR2 gene;

(3) build plasmid: with SfiI restriction enzyme respectively enzyme cut carrier pPR3-N and attR1-Cm ^r-ccdB-attR2 gene order, electrophoresis electrophoresis runs glue and reclaims, and connects and obtains pPR3-gateway carrier; By described pPR3-gateway carrier conversion ccdB survival strain competent cell, on the LB flat board containing Amp and Cm, obtain restructuring bacterium colony, check order and identify with primer pPR3-NF and pPR3-N amplification; Described pPR3-gateway carrier is transformed to DH5 α competent cell, identify lethal gene ccdB expression according to transformant production.

The present invention further provides the application of the above-mentioned plasmid vector that can be used for construction cDNA library screening, described pPR3-gateway carrier is for carrying the object carrier of goal gene by Gateway technique construction.

Preferably, described Gateway technology comprises the following steps:

A, goal gene is carried out to BP reaction, be building up on pDONOR221 carrier;

B, described pDONOR221 carrier and pPR3-gateway carrier are carried out to LR react, goal gene is fused on pPR3-gateway carrier, build the object carrier that obtains pPR3-gateway and carry goal gene.

The present invention further provides the application of the above-mentioned plasmid vector that can be used for construction cDNA library screening, above-mentioned pPR3-gateway carrier is for directed cloning, library construction and the order-checking of full-length cDNA, or for the library screening of yeast two-hybrid.

The present invention overcomes the deficiencies in the prior art, a kind of plasmid vector and construction process and application that can be used for construction cDNA library screening is provided, in the present invention, it is the membranin yeast two-hybrid system that utilizes ubiquitin mediation that membranin and host make effective means mutually, and the comparatively simple and efficient method in the construction cDNA library efficient library construction system that is Gateway.The present invention, by membranin yeast two-hybrid system and Gateway system are merged mutually, take carrier pPR3-N as template, cuts the method for connection and introduces attR1-Cm with enzyme ^r-ccdB-attR2 gene, be transformed into the pPR3-gateway vector plasmid in available Gateway technique to high-efficiency construction cDNA library, this pPR3-gateway carrier is for directed cloning, library construction and the order-checking of full-length cDNA, or for the library screening of yeast two-hybrid.

Accompanying drawing explanation

Fig. 1 is the plasmid map of pDEST22 carrier in the embodiment of the present invention 1;

Fig. 2 is that in the embodiment of the present invention 1, pPR3-Gateway(is pPR3-N-R1R2) plasmid map of carrier;

Fig. 3 is the structure schematic diagram of pPR3-Gateway carrier in the embodiment of the present invention 1;

Fig. 4 is that in the embodiment of the present invention 1, pPR3-N enzyme is cut product and Insert Fragment PCR product electrophorogram;

Fig. 5 is that the embodiment of the present invention 3 middle plateforms are identified storage capacity spirogram;

Fig. 6 is the embodiment of the present invention 3 Chinese library Insert Fragment qualification result schematic diagram.

Embodiment

In order to make object of the present invention, technical scheme and advantage clearer, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not intended to limit the present invention.

The efficient yeast vector of embodiment 1 builds

1, design of primers

As shown in Figure 1, according to plasmid map design primer attR1sfiA and the attR2sfiB of pDEST22 carrier, primer sequence is as follows for the plasmid map of pDEST22 carrier:

attR1sfiA:

5’-agtggccattacggccACAAGTTTGTACAAAAAAGCTGAACGAGAAACG-3’，

attR2sfiB:

5’-agaggccgaggcggccACCACTTTGTACAAGAAAGCTGAACGA-3’；

AttR1sfiA and attR2sfiB primer 5 ' end band SfiI restriction enzyme site,

According to upper two SfiI that intermediate sequence is different of pPR3-N, called after SfiA and SfiB respectively.

The exactness of identifying Insert Fragment sequence with pPR3-N carrier universal sequencing primer thing pPR3-NF and pPR3-NR order-checking, wherein, pPR3-NF and pPR3-NR primer sequence are as follows:

pPR3-NF：5’-GTCGAAAATTCAAGACAAGG-3’，

pPR3-NR：5’-AAGCGTGACATAACTAATTAC-3’。

2, goal gene fragment clone

Amplify the sequence (attR1-Cm between attR1 and attR2 by attR1sfiA and attR2sfiB primer from pDEST22 plasmid vector ^r-ccdB-attR2)

PCR reaction system:

On Thermal Cycler PCR instrument, carry out PCR reaction by following condition.

1%Agarose gel electrophoresis is identified order-checking, wherein, attR1 end primer band SfiA, attR2 end band SfiB, attR1 sequence as shown in SEQ ID NO.1, described Cm ^rsequence is as shown in SEQ ID NO.2, and described ccdB sequence is as shown in SEQ ID NO.3, and described attR2 sequence is as shown in SEQ ID NO.4.

3, the structure of PR3-Gateway plasmid vector

As shown in Figure 3, Fig. 3 is the structure schematic diagram of PR3-Gateway carrier, specifically comprises:

(1) cut after the PCR product (attR1-Cmr-ccdB-attR2) that pPR3-N and above primer expand by SfiI enzyme; electrophoresis run glue reclaim, electrophoresis result as shown in Figure 4, wherein; swimming lane MW molecular weight standard, swimming lane 1～2 is cut product fragment electrophoresis result for pPR3-NSfiI enzyme; Swimming lane 3～4 Insert Fragments (attR1-Cmr-ccdB-attR2) electrophoresis result.

Endonuclease reaction system:

(2) carry out ligation, ligation system:

16 ℃ of connections are spent the night, and obtain PR3-Gateway carrier, as shown in Figure 2, this PR3-Gateway carrier are transformed to ccdB survival strain competent cell.Conversion process is as follows:

A, will connect product, all be added in 100 μ l competent cells ice bath 30min;

B, heat shock 60 seconds in 42 ℃ of water-baths, transfer to cooled on ice 2min immediately;

C, add LB nutrient solution (every liter containing 10gTryptone, 5g Yeast Extract, 10g NaCl, pH7.0) the 890 μ L of ice precooling, 37 ℃ of shaking table low speed (<200rm) are cultivated 1h;

D, get 100 μ l and coat the LB flat board containing Amp and Cm, 37 ℃ of incubated overnight (10-14h).

4, plasmid identification

Carrier transformed bacteria liquid (ccdB survival strain) is coated on the LB flat board containing Amp and Cm, if normal growth shows that Insert Fragment is normal; Correct cloned plasmids transforms DH5 α competent cell, produces without transformant, shows lethal gene in Insert Fragment (ccdB) normal expression.

Embodiment 2

According to Gateway clone technology carrier construction, use Auele Specific Primer from genome or the carrier that contains object fragment, amplification object segment, PCR product electrophoresis is confirmed after band, with QIAquick Gel Extraction Kit(Qiagen) cut glue recovery, then use bP ClonaseII Enzyme Mix(Invitrogen) carry out BP reaction, be building up on pDONOR221 carrier.

BP reaction system is as follows:

In centrifuge tube, add att-PCR product	1.5ul
		PDONOR221 carrier	0.5ul
BP ClonaseII Enzyme Mix	0.5ul

Mix, 25 ℃ are incubated 1 hour, then add the Proteinase K of 0.5ul2ug/ul, hatch 10min for 37 ℃.Thermal shock transforms intestinal bacteria, the screening of Kan+ microbiotic plate, and picking mono-clonal, shakes bacterium, and upgrading grain, sends to order-checking.After order-checking is correct, enzyme is cut, and linear carrier is reclaimed in rubber tapping, then uses

lR ClonaseII Enzyme Mix(Invitrogen) carry out LR reaction, be fused in pPR3-Gateway terminals and go.

LR reaction system is as follows:

In centrifuge tube, add linear carrier	1.5ul
		PPR3-Gateway carrier	0.5ul
LR ClonaseII Enzyme Mix	0.5ul

Mix, 25 ℃ are incubated 1 hour, then add the Proteinase K of 0.5ul2ug/ul, hatch 10min for 37 ℃.Thermal shock transforms intestinal bacteria, microbiotic plate screening (determining according to concrete carrier), and picking mono-clonal, shakes bacterium, upgrading grain, sequence verification exactness.

The efficient yeast two-hybrid library construction of embodiment 3

1, the extraction of TotalRNA

Carry out with reference to Trizol specification sheets.Get 100mg tissue sample, use liquid nitrogen grinding powder in the mortar of precooling, then proceed in 1.5ml centrifuge tube, add the Trizol reagent of 1ml, vibration mixes, and room temperature leaves standstill the abundant lysing cell of 10min; Add the chloroform of 200ul, concuss 15sec, room temperature leaves standstill 5min; At 4 ℃, 12,000 × g high speed frozen centrifugation 15min; Get supernatant in new 1.5ml centrifuge tube, add 0.5ml Virahol, put upside down and mix, room temperature leaves standstill 10min; At 4 ℃, 12,000 × g high speed frozen centrifugation 10min; Remove supernatant, add the 75% washing with alcohol precipitation of 1ml, 7,500 × g high speed frozen centrifugation 5min(repeats this step once); Remove supernatant, air drying RNA precipitation, is then dissolved in 50ulDEPC water.

2, the separation of mRNA

Reference

2.0Kit specification sheets carries out.

(1) by the Total RNA precipitation of > 500ug, be resuspended in the DEPC water of 100ul.

(2) get new 50ml centrifuge tube, add the Stock Buffer of 10ml and the ProteinaseK of 200ul, form the Lysis Buffer that mRNA separates, then the Total RNA of upper step is added in Lysis Buffer, put upside down and mix.

(3) 65 ℃ of water-bath 5min, then put 1min on ice immediately.

(4) add the 5MNaCl of 650ul, put upside down and mix.

(5) get a pipe oligo(dT) cellulose, add in above-mentioned buffer, put upside down and mix 2min.

(6) 50ml centrifuge tube is placed in to mixed at room temperature 60min on mixed instrument.

(7) room temperature, the centrifugal 5min of 3,000 × g, removes supernatant.

(8) add the Binding Buffer of 20ml, resuspended precipitation, room temperature, the centrifugal 5min of 3,000 × g, removes supernatant.

(9) add the Binding Buffer of 10ml, resuspended precipitation, room temperature, the centrifugal 5min of 3,000 × g, removes supernatant.

(10) add the Low Salt Wash Buffer of 10ml, resuspended precipitation, room temperature, the centrifugal 5min of 3,000 × g, removes supernatant; Repeat this step 4 time.

(11) precipitation is resuspended in to the Low Salt Wash Buffer of 800ul, and forwards in spin-column, then spin-column is placed in to collection tube, room temperature, the centrifugal 10sec of 5000 × g, removes filtered solution

(12) add the Low Salt Wash Buffer washing precipitation of 500ul, room temperature, the centrifugal 10sec of 5000 × g, removes filtered solution; Repeat this step 4 time.

(13) spin-column is placed in to new 2ml centrifuge tube, adds the Elution Buffer of 200ul, abundant resuspended precipitation, room temperature, the centrifugal 30sec of 5000 × g, must guard against and do not remove elutriant; Add again the Elution Buffer of 200ul, abundant resuspended precipitation, room temperature, the centrifugal 30sec of 5000 × g, obtains two parts of 400ul elutriants altogether.

(14) add the 2M sodium acetate of 60ul, and 1ml dehydrated alcohol, put upside down and mix, be placed in-80 ℃ of 15min.

(15), at 4 ℃, 14000rpm high speed frozen centrifugation 15min, removes supernatant.

(16) 75% washing with alcohol precipitations, 4 ℃, 14000rpm high speed frozen centrifugation 10min, removes supernatant.

(17) in air, dry precipitation, be finally dissolved in the DEPC water of 50ul, be placed in-80 ℃ of preservations.

3, the structure of cDNA library (Uncut type)

Carry out with reference to CloneMiner specification sheets.

(1) cDNA the first chain is synthetic

A gets the mRNA3ug obtaining in step, is dissolved in 9ulDEPC water;

B adds biotin-attB2-Oligo(dT) Primer(30pmol) 1ul, and 10mM(each) dNTPs1ul, 65 ℃ of 5min, cool to 45 ℃, 2min;

C adds 5X First Strand Buffer4ul, DTT(0.1M) 2ul, 45 ℃, 2min;

D adds SuperScript III RT enzyme 3ul, 45 ℃, 60min.

(2) cDNA the second chain is synthetic

A according to the form below in above-mentioned reaction solution adds each composition:

16 ℃, 2 hours;

B adds T4DNA Polymerase2ul, and 16 ℃, 5min;

C adds 0.5M EDTA(pH8.0) 10ul;

D adds phenol: chloroform: primary isoamyl alcohol (25:24:1) 160ul, fully mixes 30sec; 14000rpm, the centrifugal 5min of room temperature, is carefully taken into supernatant in new centrifuge tube;

E according to the form below adds each composition:

Glycogen（20ug/ul） 1ul

7.5M NH ₄OAc 80ul

100%ethanol 600ul

-80 ℃, leave standstill 15min; 14000rpm, 4 ℃, centrifugal 25min, removes supernatant;

F adds 150ul70% washing with alcohol precipitation; 14000rpm, 4 ℃, centrifugal 3min, removes supernatant;

G adds 150ul70% washing with alcohol precipitation; 14000rpm, 4 ℃, centrifugal 3min, removes supernatant;

H room temperature 5-10min dries cDNA;

I blows and beats 30-40 time repeatedly with the DEPC water of 18ul, dissolves cDNA.

(3) cDNA is connected (three kinds of respectively each connections of joint are a) with three frame attB1 restructuring joints

Mix latter 16 ℃, 16-24 hour.

(4) cDNA fractional separation and collection

Fixing fractionation column, opens upper cover, then opens stopper below.Treat that in pillar, storage liquid is flow to end, add 0.8mlTEN Buffer clean pillar, determine pillar flow velocity be every 30～40 seconds one, every 25～35ul, otherwise the fractionation column that need to more renew (repeating this step 3 time); Be ready to 20 new 1.5ml centrifuge tubes, mark 1～20; The cDNA product of three parts of each 50ul is mixed and added in pillar, collect and filter product in No. 1 pipe; Again to the TEN Buffer that adds 50ul in pillar, collect and filter product and manage with No. 2; At every turn, to the TEN Buffer that adds 100ul in pillar, collect 1 filtration product since No. 3 every pipes of pipe; The cDNA output of collecting with the every pipe of spectrophotometric determination, with the pipettor cDNA volume that accurately definite every pipe is collected, the cDNA product that before merging, 150ng collects.

According to the form below adds each composition:

Glycogen（20ug/ul） 1ul

7.5M NH ₄OAc 0.5V

100%ethanol 2.5V

-80 ℃, leave standstill 15min; 14000rpm, 4 ℃, centrifugal 25min, removes supernatant; Add 150ul70% washing with alcohol precipitation; 14000rpm, 4 ℃, centrifugal 3min, removes supernatant; Add 150ul70% washing with alcohol precipitation; 14000rpm, 4 ℃, centrifugal 3min, removes supernatant; Room temperature 5～10min dries Cdna; Repeatedly blow and beat 30～40 times with the DEPC water of 9ul, dissolve cDNA.

(5) BP recombining reaction

According to the form below adds each composition:

Mix and be placed on 25 ℃, 16～20 hours.

(6) electricity transforms intestinal bacteria DH10B

In BP recombining reaction system, add the ProreinaseK of 2ul, 37 ℃, 15min; 75 ℃, 10min, according to the form below adds each composition:

-80 ℃, leave standstill 15min; 14000rpm, 4 ℃, centrifugal 25min, removes supernatant; Add 150ul70% washing with alcohol precipitation; 14000rpm, 4 ℃, centrifugal 3min, removes supernatant; Add 150ul70% washing with alcohol precipitation; 14000rpm, 4 ℃, centrifugal 3min, removes supernatant; Room temperature 5～10min dries cDNA; Repeatedly blow and beat 30～40 times with the TE Buffer of 8ul, dissolve cDNA; First by the revolving cup-80 ℃ precooling of 1mm electricity, on ice, 2ul recombinant products and 50ul electricity are turned to competent cell DH10B and add electric revolving cup, put 45min on ice; After electric shock, in electric revolving cup, add SOC substratum 1ml rapidly; Point is turned to thing and be taken into new 15ml centrifuge tube, be placed in 37 ℃, 225～250rpm shaking table is cultivated 1 hour; After cultivation finishes, identify for storage capacity according to 2.4.1 method spread plate; Residue culture adds glycerine to final concentration 20% to be stored in-80 ℃, and this is library bacterium liquid.

(7) cDNA library (Uncut type) library quality evalution

The evaluation of A storage capacity

Get transform after after 1000 times of bacterium stoste (1ml) 10ul dilutions, therefrom take out 50ul coating LB flat board (containing corresponding resistant), second day counting.

Clone number/50ul × 1000 on total clone's number CFU=flat board times × 1 × 10 ³ul

B recombination fraction and Insert Fragment length are identified

Random 24 clones of picking carry out bacterium colony PCR evaluation

In Microtube, be formulated as follows reaction solution:

1%Agarose gel electrophoresis is identified.

4, the plasmid extraction in Uncut library

Upper step is demonstrate,proved to qualified Uncut library, adopt broth culture, shake bacterium, 30 ℃ of incubated overnight, took out plasmid in second day, and plasmid is surveyed OD electrophoresis detection.

5, library plasmid restructuring

By in take out the plasmid obtaining, be diluted to 300ng/ul, according to following Establishing LR recombining reaction, according to the form below adds each composition:

Mix and be placed on 25 ℃, 16～20 hours, 2.6.3 electricity transformed intestinal bacteria DH10B.In LR recombining reaction system, add the ProreinaseK of 2ul, 37 ℃, 15min; 75 ℃, 10min, according to the form below adds each composition:

-80 ℃, leave standstill 15min; 14000rpm, 4 ℃, centrifugal 25min, removes supernatant; Add 150ul70% washing with alcohol precipitation; 14000rpm, 4 ℃, centrifugal 3min, removes supernatant; Add 150ul70% washing with alcohol precipitation; 14000rpm, 4 ℃, centrifugal 3min, removes supernatant; Room temperature 5～10min dries Cdna; Repeatedly blow and beat 30～40 times with the TEBuffer of 8ul, dissolve cDNA; First by the revolving cup-80 ℃ precooling of 1mm electricity, on ice, 2ul recombinant products and 50ul electricity are turned to competent cell DH10B and add electric revolving cup, put 45min on ice; After electric shock, in electric revolving cup, add SOC substratum 1ml rapidly; Point is turned to thing and be taken into new 15ml centrifuge tube, be placed in 37 ℃, 225～250rpm shaking table is cultivated 1 hour; After cultivation finishes, identify for storage capacity according to 2.4.1 method spread plate; Residue culture adds glycerine to final concentration 20% to be stored in-80 ℃, and this is library bacterium liquid.

6, Yeast two-hybrid cDNA library quality evalution

(1) evaluation of storage capacity

Get transform after after 1000 times of bacterium stoste (1ml) 10ul dilutions, therefrom take out 50ul coating LB flat board (containing corresponding resistant), second day counting, as shown in Figure 5, Fig. 5 is plate assay storage capacity spirogram.

Clone number/50ul × 1000 on total clone's number CFU=flat board times × 1 × 10 ³ul.

Clone number/0.5ul × 1000 on flat board times=850/0.5ul × 1000 times=1.7 × 10 ⁶cfu/ml

Total clone number CFU=1.7 × 10 ⁶cfu/ml × 4ml=0.68 × 10 ⁷.

(2) recombination fraction and Insert Fragment length are identified

Random 36 clones of picking carry out bacterium colony PCR evaluation

In Microtube, be formulated as follows reaction solution:

On Thermal Cycler PCR instrument, carry out PCR reaction by following condition:

The evaluation of 1%Agarose gel electrophoresis, as shown in Figure 6, Fig. 6 is library Insert Fragment qualification result schematic diagram; Wherein, M:1kb DNA ladder; Swimming lane 1～32 is cDNA Insert Fragment.

Compare and the shortcoming and defect of prior art, the present invention has following beneficial effect: the present invention obtains the gateway carrier of an efficient quick, can be used for directed cloning, library construction and the order-checking of full-length cDNA; Be a Yeast expression carrier simultaneously, can be used as the plasmid in the screening library of yeast two-hybrid.

The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any modifications of doing within the spirit and principles in the present invention, be equal to and replace and improvement etc., within all should being included in protection scope of the present invention.

Claims

1. A plasmid vector that can be used to construct cDNA library screening is characterized in that it is composed of pPR3-N carrier and attR1-Cm ^r -ccdB-attR2 gene, and is denoted as pPR3-gateway carrier, wherein the attR1 sequence is as follows As shown in SEQ ID NO.1, the Cm ^r sequence is shown in SEQ ID NO.2, the ccdB sequence is shown in SEQ ID NO.3, and the attR2 sequence is shown in SEQ ID NO.4.

2. the plasmid vector that can be used for constructing cDNA library screening as claimed in claim 1, is characterized in that, the 5' end and the 3' end of described attR1-Cm ^r- ccdB-attR2 gene are subjected to SfiI restriction endonuclease respectively The enzyme cut point is connected to the pPR3-N vector.

3. The plasmid vector capable of constructing cDNA library screening according to claim 2, wherein the attR1-Cm ^r -ccdB-attR2 gene is the sequence between attR1 and attR2 on the vector pDEST22.

4. A construction method that can be used to construct a plasmid vector for cDNA library screening, is characterized in that comprising the following steps:

(1) Design primers attR1sfiA and attR2sfiB for the sequence between attR1 and attR2 on the vector pDEST22, and select pPR3-N vector general sequencing primers pPR3-NF and pPR3-NR; wherein, the sequences of primers attR1sfiA and attR2sfiB are as follows:

attR1sfiA:

5'-agtggccattacggccACAAGTTTGTACAAAAAAGCTGAACGAGAAACG-3',

attR2sfiB:

5'-agaggccgaggcggccACCACTTTGTACAAGAAAGCTGAACGA-3';

The pPR3-NF and pPR3-NR primer sequences are as follows:

pPR3-NF: 5'GTCGAAAATTCAAGACAAGG3',

pPR3-NR: 5'AAGCGTGACATAACTAATTAC3';

(2) Amplified sequence: using the DNA sequence of the vector pDEST22 as a template, the attR1-Cm ^r -ccdB-attR2 gene was amplified with primers attR1sfiA and attR2sfiB;

(3) Plasmid construction: SfiI restriction endonuclease was used to digest the vector pPR3-N and attR1-Cm ^r -ccdB-attR2 gene sequences respectively, and the gel was recovered by electrophoresis and electrophoresis, and the pPR3-gateway vector was obtained by ligation; the pPR3 -gateway vector to transform ccdB survival strain competent cells, obtain recombinant colonies on LB plates containing Amp and Cm, use primers pPR3-NF and pPR3-N to amplify and sequence for identification; transform the pPR3-gateway vector into DH5α competent cells cells, and identify the expression of the lethal gene ccdB according to the production of transformants.

5. The application of the plasmid vector capable of constructing cDNA library screening according to any one of claims 1 to 3, characterized in that, the pPR3-gateway vector is used to construct a target vector carrying a target gene through Gateway technology.

6. the application that can be used for constructing the plasmid vector of cDNA library screening as claimed in claim 5, is characterized in that, described Gateway technology comprises the following steps:

A. Perform BP reaction on the target gene and construct it on the pDONOR221 vector;

B. Perform LR reaction between the pDONOR221 vector and the pPR3-gateway vector, fuse the target gene to the pPR3-gateway vector, and construct the target vector carrying the target gene in pPR3-gateway.

7. The application of the plasmid vector that can be used to construct cDNA library screening according to any one of claims 1 to 3, characterized in that the pPR3-gateway vector according to any one of claims 1 to 3 is used for full-length cDNA directional cloning, library construction and sequencing, or library screening for yeast two-hybrid.