CN108841866A

CN108841866A - A kind of adenovirus vector and its construction method

Info

Publication number: CN108841866A
Application number: CN201810565182.5A
Authority: CN
Inventors: 蓝田; 罗燕; 施金秀; 王焕荣
Original assignee: Yun Zhou Biotechnology (guangzhou) Co Ltd
Current assignee: Yun Zhou Biotechnology (guangzhou) Co Ltd
Priority date: 2018-06-04
Filing date: 2018-06-04
Publication date: 2018-11-20
Anticipated expiration: 2038-06-04
Also published as: CN108841866B

Abstract

The invention discloses provide a kind of recombinant adenoviral vector and its construction method.This method is to go to pRed/ET (Δ α) electricity in the DB3.1 competence containing pAV.Des1d carrier, homologous recombination is carried out with the Kan resistance fragments comprising homology arm, only there is Amp+Kan resistance by filtering out under continuous resistance pressure, without the positive colony of Amp+Cm resistance.Second step recombination is recombinated with the attR4-Cm-ccdB-attR2 with homology arm or attR4-Cm-ccdB-attR3, recycling resistance screening to go out only has Amp+Cm resistance, without the correct positive colony of Amp+Kan resistance, to obtain adenovirus vector of the present invention.Adenovirus expression carrier of the present invention, and there is good accuracy, high efficiency, success rate is high.Human time's cost is greatly reduced, user experience is improved.

Description

A kind of adenovirus vector and its construction method

Technical field

The invention belongs to field of biotechnology, it is related to a kind of adenovirus vector and preparation method thereof.

Background technique

Adenovirus is a kind of nonencapsulated linear dsdna virus, is widely present in the Nature, full-length genome is about 36kb.Adenovirus has almost 100% efficiency of infection to most cells, including division and not dividing cell and primary cell, Almost all unconformity therefore will not interfere other host's bases into chromosome in all known cells other than egg cell Cause.Due to the genome of adenoviral gene group unconformity to host, so adenovirus is transient expression in host.Adenovirus It can be commonly used in vaccine and gene therapy with direct infection animal individual.Due to the above various advantages, adenovirus vector is a kind of Widely used virus carrier system, wherein most popular is 5 type serotype adenovirus carriers.In order to improve adenopathy The biological safety of poisonous carrier, people are transformed adenoviral gene group, construct the early expression base for having lacked adenovirus Because of the recombinant adenoviral vector in the area sequence E1 and E3.E1 is necessary to adenoviral replication, and the missing of E1 keeps it multiple from itself System, the trans-complementation that can only be provided by incasing cells such as 293A cell carries out duplication amplification, without can be carried out autonomous duplication, E3 The albumen of gene expression can fight the antiviral defense system of host, and the removal in the area E3 can be reduced the humoral immune reaction of host.

Gateway technology is the general clone technology of one kind based on λ bacteriophage site specific recombination system, can be quick Allogeneic dna sequence DNA segment is cloned into different carriers by ground with high specificity.Its main feature is that reacting finally handle by BP reaction and LR Target gene is cloned on expression vector.Recombination is played in Gateway technology is recombination site：attB,attP, AttL and attR.In BP Clonase^TMII enzyme mix catalysis recombining reaction in, the site attB usually with the site attP It recombinates.BP recombining reaction (BP recombination) refers to the donor vehicle (donor vector) containing the site attP Att site sequence exchange reaction between the PCR product containing the site attB or other clones.Donor vehicle, which carries, blocks that Mycin resistant gene, chloramphenicol resistance gene and a ccdB suicide gene.Chloramphenicol resistance gene and ccdB suicide gene position Between two sites attP.Since ccdB suicide gene can be only present in ccdB T1surviral cells, ccdB T2 In surviral cells or DB3.1, and there is lethal effect to these cells of Top10, stbl3, DH5a.It is supplied to duplication Body carrier, it is necessary to donor vehicle be transformed into ccdB T1surviral cells, ccdB T2 surviral cells or It could be replicated as host divides in DB3.1.BP recombining reaction the result is that the aim sequence in attB-PCR product exchanges Into the donor vehicle containing the site attP, and the ccdB suicide gene and chloramphenicol resistance gene in donor vehicle are swapped out It goes.BP recombining reaction product is transformed into non-ccdB surviral cells, such as Top10, then with LB containing kanamycin Plate screening positive colony.Double Selection based on kanamycins and suicide gene, 90% clone are correctly to clone. After the recombination of the site attB and attP, sequence is exchanged, the attP on the donor vehicle containing aim sequence after leading to recombination Site sequence produces variation, and the site attP becomes into the site attL.The donor vehicle after recombination is generally known as entry clones (entry clone)。

LR recombining reaction (LR recombination) refers to the entry clones containing the site attL and contains the site attR Purpose carrier (destination vector) between att site sequence exchange reaction.Purpose carrier carries ampicillin Resistant gene, chloramphenicol resistance gene and ccdB gene.Chloramphenicol resistance gene and ccdB suicide gene be located at the site attR it Between.Due to ccdB suicide gene can be only present in ccdB T1surviral cells, ccdB T2 surviral cells or In DB3.1, and there is lethal effect to these cells of Top10, stbl3, DH5a.To replicate purpose carrier, it is necessary to purpose Carrier be transformed into ccdB T1surviral cells or ccdB T2 surviral cells could with host divide and Duplication.LR recombining reaction the result is that the aim sequence between the site entry clones attL exchange to the purpose containing the site attR load In vivo, and ccdB suicide gene and chloramphenicol resistance gene are swapped out.LR reaction product is transformed into non-ccdB In surviral cells, such as stbl3, then with the LB plate screening positive colony containing ampicillin.Based on ampicillin and The Double Selection of suicide gene, 90% clone are correctly to clone.After the recombination of the site attL and attR, sequence is handed over It changes, the attR site sequence on the purpose carrier containing aim sequence after leading to recombination produces variation, reassembles into attB Point sequence.Such purpose carrier is generally known as expression cloning or expression vector (expression vector). Gateway system type has following three kinds：

1) single slice recombination system (a fragment recombination system)

In single slice recombination system, one section of end contain the PCR product of attB1 and attB2 and containing attP1 and BP reaction occurs for the donor vehicle pDONR 221 of attP2, generates the entry clones pDown-xxx containing aim sequence.This enters Door clone then occurs LR with the purpose carrier containing attR1 and attR2 and reacts, and generates the expression containing one section of aim sequence and carries Body.

2) biplate section recombination system (two fragments recombination system)

In biplate section recombination system (such as Fig. 1), PCR product and contain that attB4 and attB1r is contained in one section of end BP reaction occurs for the donor vehicle pDONR P4P1R of attP4 and attP1r, generates the entry clones pUp- containing aim sequence xxx.Contain the PCR product of attB1 and attB2 and the donor vehicle pDONR 221 containing attP1 and attP2 in another section of end BP reaction occurs and generates pDown-xxx.Then with the purpose carrier containing attR4 and attR2 LR occurs for the two entry clones Reaction generates the expression vector containing two sections of aim sequences.

3) three segment recombination system

In three segment recombination systems, one section of end contain the PCR product of attB4 and attB1r and containing attP4 and The donor vehicle pDONRP4P1r of attP1r occurs BP reaction and generates pUp-xxx, and attB1's and attB2 contained in another section of end PCR product and donor vehicle pDONR 221 containing attP1 and attP2 occur BP and react pDown-xxx, and third section end contains There are the PCR product of attB2r and attB3 and the donor vehicle pDONR P2rP3 containing attP2r and attP3 that BP reaction occurs to produce Raw pTail-xxx.These three entry clones then occur LR with the purpose carrier containing attR4 and attR3 and react, and generation contains The expression vector of three sections of aim sequences.

Red/ET homologous recombination technique is a kind of based on Rac bacteriophage RecE/RecT or λ bacteriophage Red α/Red β/Red DNA homologous recombination system in the Escherichia coli body of γ, by homology arm, under the action of homologous recombination enzyme, can by purpose Segment homologous recombination is to selection area, to generate specific site missing, insertion, the point mutation etc. that researcher wants.pRed/ET It is a kind of homologous recombination expression of enzymes carrier of responsive to temperature type, the pBAD promoter comprising the induction of a L-arabinose, entirely Homologous recombination process is mainly cooperateed with completion by three kinds of albumen.Wherein there are 5 ' -3 ' nucleic acid from the Red α albumen of λ bacteriophage 5 prime excision enzyme activity；Red β is a kind of annealing albumen, both albumen synergistic effect is catalyzed entire homologous recombination；In addition λ is derived from The Red γ albumen of bacteriophage can be in conjunction with the RecBCD exonuclease of endogenous E. coli.

Conventional carrier of the recombinant adenoviral vector as a kind of bigger (~36kb), utilizes conventional digestion connection method Seem on operating level gruelling, facing a problem is after large fragment and small fragment are cut, and large fragment is difficult to recycle, city Plastic recovery kit recycling segment on face is all 10kb hereinafter, being therefore difficult effectively to recycle skeleton carrier.Big portion Single cent offers the method for being related to being all based on homologous recombination to the transformation of adenoviral backbone, such as wherein document utilization Escherichia coli Host strain of the BJ5183 recBC sbcBC as recombination.

Existing recombinant adenoviral vector is the flexible and efficient cloning vector of one kind based on gateway clone technology PAV.Des1d (Invitrogen company commodity), Vector map is as shown in Figure 2.The carrier remains wild-type adenovirus load The region ITR at body both ends and most genomes, have lacked the area E1 and E3, there are one gateway cloning site, structures For attR1-Cm-ccdB-attR2, as shown in Fig. 2, since ccdB suicide gene can be only present in ccdB T1surviral In cells, ccdB T2 surviral cells or DB3.1, so the carrier needs to be transformed into one such host strain In replicated.According to demand, first aim sequence can be cloned into pDONR carrier by BP reaction and forms entry clones, so LR reaction is carried out again afterwards to be cloned on adenovirus vector pAV.Des1d.But the carrier belongs to the list in gateway carrier system Segment recombination system if it is multiple clips clone then needs multiple clips to merge to get up to be cloned into ability in pDONR carrier Carry out subsequent work.Multiple clips fusion DNA vaccine tends to occur merging unsuccessful situation, not only increase consumables cost and As a result time cost cannot guarantee that, what this was detrimental to efficiently to produce.

In brief, existing adenovirus vector and its construction method have the following disadvantages：First, existing adenovirus vector packet AttR1-attR2 recombination site is contained, so a LR reaction can only clone the external source purpose piece from entry clones Section is then needed these segment compositions with the method for fusion DNA vaccine if necessary to clone multiple exogenous sequences simultaneously into a piece Section, then subsequent building is carried out, this not only increases the difficulty of building, also increases time and human cost, is unfavorable for improving Production efficiency.Second, after skeleton digestion, it is difficult to carry out gel extraction with existing plastic recovery kit on the market, even if recycling, Whether can to skeleton segment cause mechanical damage, lead to fragment loss if cannot guarantee that.Third, traditional Red/ET homologous recombination Unexpected intramolecular rearrangement, the probability of dystopy of technology appearance are very high, and the probability for false positive occur is high, real positive rate It is low.

Summary of the invention

In order to solve above-mentioned technical problem, this application provides a kind of recombinant adenoviral vector and its building sides Method.As long as so that gained adenovirus vector carry out a step LR reaction, can by multiple segments simultaneously fast and flexible efficiently clone Into adenovirus vector, substantially reduces the seeervice cycle and saved time consumables cost, improve synthesized competitiveness.And it can Positive rate has been increased to about 50% from original 1%.

The object of the present invention is to provide a kind of recombinant adenoviral vector and its construction methods.

The technical solution adopted by the present invention is that：

A kind of construction method of adenovirus vector, includes the following steps：

1) by the Red α gene elmination in pRed/ET carrier, pRed/ET (Δ α) carrier is obtained；

2) pRed/ET (Δ α) carrier is transferred to the DB3.1 competent cell of the plasmid containing pAV.Des1d, recovery culture；

3) product after recovery culture is applied to simultaneously containing ammonia benzyl antibiotic Amp, chloramphenicol Cm and tetracycline Tc On culture medium flat plate, it is protected from light culture, picking positive colony bacterium A；

4) simultaneously by inducing expression homologous recombination enzyme in culture medium of the addition of positive colony bacterium A obtained by upper step containing L-arabinose It is prepared into competent cell, Kan resistance expression's box PCR product with homology arm is transferred to and wherein carries out recombining reaction, recovery training It supports；

5) the recovery cultured products of step 4) are applied on the culture medium flat plate containing Amp, Kan and Tc, are protected from light culture, chosen Take recombinant clone bacterium B；

6) the difference liquid medium within of positive colony bacterium B obtained in step 5) is expanded into culture, wherein containing Amp, Kan The clone bacterium that grows in the resistance culture base of Tc, and cannot grow in the base of resistance culture containing Cm is purpose positive colony bacterium C；

7) L-arabinose inducing expression homologous recombination enzyme is added in purpose positive colony bacterium C obtained in step 6) and made It is standby at competent cell, by the attR4-Cm-ccdB-attR2 with homology arm or with the attR4-Cm-ccdB-attR3 of homology arm Segment, which is transferred to, wherein carries out recombining reaction；Recovery culture；

8) recovery cultured products in step 7) are applied on the culture medium flat plate containing Amp, Cm and Tc, 36.5~37.5 DEG C Culture, picking recombinant clone bacterium D；

9) the difference liquid medium within of positive colony bacterium D obtained in step 8) is expanded into culture, wherein containing Amp and Cm Resistance culture base in grow, and the clone bacterium that cannot be grown in containing Kan and Tc resistance culture base is purpose positive colony Bacterium E；The cultivation temperature of all cultures is 36.5~37.5 DEG C in the step；

The plasmid in positive colony bacterium E is extracted up to adenovirus vector.

Further, also Red γ and Red beta gene sequence have been carried out in step 1), in pRed/ET (Δ α) carrier excellent Change, Red γ and the Red beta gene sequence after optimization are respectively such as SEQ ID NO:6,SEQ ID NO:Shown in 7.

Further, step 1)~7) in all cultures cultivation temperature be 29.5~30.5 DEG C.

Further, the base sequence such as SEQ ID NO of the Kan resistance fragments with homology arm:Shown in 1.

Further, the base sequence of the attR4-Cm-ccdB-attR2 with homology arm such as SEQ ID NO:2 institutes Show.

Further, the base sequence of the attR4-Cm-ccdB-attR3 with homology arm such as SEQ ID NO:3 institutes Show.

Further, in step 2), recovery culture culture medium used is SOC culture medium.

The adenovirus vector of any of the above-described the method building.

Thallus containing adenovirus vector described above.

A kind of adenovirus vector pAV.Des2d, compared with existing pAV.Des1d carrier, distinctive points are, PAV.Des2d is with attR4 instead of the attR1 sequence in pAV.Des1d, while pAV.Des2d is connected with termination in the downstream attR2 Subsequence.

A kind of adenovirus vector pAV.Des3d, compared with existing pAV.Des1d carrier, distinctive points are, PAV.Des3d with attR4 instead of the attR1 sequence in pAV.Des1d, while with attR3 instead of in pAV.Des1d AttR2 sequence, and pAV.Des3d is connected with terminator sequence in the downstream attR3.

The beneficial effects of the invention are as follows：

(1) the method for the present invention, which needs not move through, recombines, can be by multiple clips Gateway recombination site and adenovirus vector Combination, building obtain pAV.Des2d and pAV.Des3d, prevent the commercial monopoly of pAV.Des1d.And feel in the present invention in DB3.1 By progress homologous recombination reaction in state (anti-ccdB suicide gene).

(2) pAV.Des2d the and pAV.Des3d adenovirus vector that the present invention obtains realizes same on adenovirus vector Shi Kelong multiple clips.

(3) recombining reaction that the present invention is mediated with new pRed/ET (Δ α), although the colony counts on plate reduce, But its positive rate has been increased to about 50% from original 1%, the accuracy of positive colony greatly reduces vacation close to 100% The generation of positive colony.This also solves us and endures the second step recombination positive rate (weight in 1 step 7 of embodiment to the fullest extent all the time Group reaction) extremely low puzzlement, it improves work efficiency.In actual operation, we are by relatively popular promoter, purpose base Cause and tracer gene etc., which are building up to respectively in corresponding pDONR carrier, forms corresponding entry clones, then not according to client With needing to carry out flexibly freely arrange in pairs or groups, as long as one step LR of progress reacts, can by multiple segments and meanwhile fast and flexible efficiently gram It is grand into adenovirus vector, substantially reduce and the seeervice cycle and saved time consumables cost, the synthesis for improving our company is competing Strive power.

(4) the existing recombinant adenoviral vector of the applicant is that a kind of high-efficient cloning based on gateway recombination system carries Body, but since the carrier contains attR1-attR2 recombination site, so a LR reaction can only clone one from entering The external source target fragment of door clone, if client needs while cloning multiple exogenous sequences, needing will with the method for fusion DNA vaccine These segment compositions are at a segment, then carry out subsequent building, this not only increases the difficulty of building, also increase the time and Human cost is unfavorable for us and improves production efficiency.In order to improve clone's applicability of the carrier, flexibility, on this basis, We are transformed existing recombinant adenoviral vector by specific method, with attR4-attR2 and attR4-attR3 points Not Qu Dai attR1-attR2, construct the recombined adhenovirus cloning vector that can clone 2 and 3 target fragments simultaneously PAV.Des2d and pAV.Des3d, so that the building of recombinant adenoviral expressing vector becomes high efficient and flexible.

Detailed description of the invention

Fig. 1 biplate section recombination system；

Fig. 2 is pAV.Des1d Vector map；

Fig. 3 is pRed/ET (Δ α) Vector map；

Fig. 4 is pAV.Des2d Vector map；

Fig. 5 is pAV.Des3d Vector map；

Fig. 6 is the flow diagram of adenovirus vector construct method of the present invention；

Fig. 7 is by positive expression carrier pAV [Exp]-CMV>EGFP transfects 293A cell, and obvious lesion spot is found after 7 days, Adenovirus is packed in success；Left figure：The white light visual field；Right figure：The green fluorescence visual field；

Fig. 8 is positive expression carrier pAV [Exp]-CMV>EGFP:IRES:MCherry transfects 293A cell, finds after 7 days Obvious lesion spot, successfully packs adenovirus；Left figure：The white light visual field；Middle figure：The green fluorescence visual field；Right figure：The red fluorescence visual field.

Specific embodiment

The present invention is further illustrated combined with specific embodiments below.

A kind of construction method of the adenovirus vector of embodiment 1

1. the Red α gene elmination in pRed/ET carrier (being purchased from biotech firm) is optimized Red β and Red γ gene sequence Column, obtain pRed/ET (Δ α) carrier of Optimizing Reconstruction, recombinate the non-specific large fragment intramolecular generated in regrouping process Rate significantly reduces, to achieve the purpose that improve specific recombination efficiency.The structural representation of the pRed/ET (Δ α) of Optimizing Reconstruction Scheme the core sequence such as SEQ ID NO as shown in figure 3, in pRed/ET (Δ α) carrier:Shown in 4, including pBAD promoter Sequence (SEQ ID NO:5) Red gamma gene sequences (the SEQ ID NO after, optimizing:6) the Red beta gene sequence (SEQ after, optimizing ID NO:7).PRed/ET (Δ α) electricity of optimization is gone in the DB3.1 competent cell comprising pAV.Des1d, SOC training is added It supports and is cultivated 1 hour based on 30 DEG C of recoveries.

2. recovery cultured products are applied to simultaneously containing ammonia benzyl antibiotic (Amp), chloramphenicol (Cm) and tetracycline (Tc) On LB plate, 30 DEG C are protected from light (tetracycline decomposes in light) and cultivate two days, choose clone PCR and identify the positive gram containing two kinds of plasmids It is grand.

3. picking positive colony is inoculated into the LB liquid medium of the resistance containing Amp+Cm+Tc respectively and is incubated overnight for 30 DEG C.

4. L-arabinose inducing expression homologous recombination enzyme is added in positive colony bacterium obtained in step 3 and is prepared into sense By state cell, by Kan resistance fragments (the base sequence such as SEQ ID NO with homology arm:Shown in 1) PCR product electricity is transferred to wherein Carry out recombining reaction.

5. recovery cultured products are applied on the LB plate of Amp+Kan+Tc, 30 DEG C are protected from light (tetracycline decomposes in light) culture two It, chooses clone PCR and identifies recombinant clone.

It is incubated overnight 6. positive colony obtained in step 5 is inoculated into respectively in LB liquid medium in 30 DEG C, wherein The clone that grows in Amp+Kan+Tc resistance culture base, and cannot grow in Cm resistance culture base is purpose positive colony

7. L-arabinose inducing expression homologous recombination enzyme is added in positive colony bacterium obtained in step 6 and is prepared into sense By state cell, by attR4-Cm-ccdB-attR2 (the SEQ ID NO with homology arm:Or attR4-Cm-ccdB-attR3 2) (SEQ ID NO:3) segment electricity, which is transferred to, wherein carries out recombining reaction.

8. recovery cultured products are applied on the LB plate of Amp+Cm+Tc, 37 DEG C are cultivated one day, are chosen clone PCR and are identified weight Group positive colony.

It is incubated overnight 9. positive colony obtained in step 8 is inoculated into respectively in LB liquid medium in 37 DEG C, wherein The clone that grows in Amp+Cm resistance culture base, and cannot grow in Kan and Tc resistance culture base is the pure positive gram Grand bacterium；The plasmid of positive colony bacterium is extracted up to adenovirus vector.When conversion with segment is the attR4- with homology arm in step 7 When Cm-ccdB-attR2 segment, final resulting adenovirus vector is denoted as pAV.Des2d, carrier schematic diagram as shown in figure 4, The corresponding Chinese of each component symbol title is as shown in table 1 in figure；When conversion with segment is with homology arm in step 7 When attR4-Cm-ccdB-attR3 segment, final resulting adenovirus vector is denoted as pAV.Des3d, carrier schematic diagram such as Fig. 5 Shown, the corresponding Chinese of each component symbol title is as shown in table 1 in figure.

The positive rate for further obtaining pAV.Des2d and pAV.Des3d to the present embodiment detects, the results show that The positive rate of pAV.Des2d and pAV.Des3d is respectively 54%, 50%, and the accuracy of positive colony is 100% (sun after verifying Property rate to refer to that bacterium on one sheet has plenty of wrong, have plenty of pair, expanded by bacterium colony PCR and see if there is purpose band, Purposeful band is then considered positive, to calculate positive rate；And positive colony accuracy refers to that positive colony have passed through into one Pacing sequence, sequence are the ratios that right-on quantity accounts for total positive colony number).And use the existing pRed/ET not being transformed Carrier (gene of α containing Red, Red β and Red gamma gene sequences are not optimised) carries out the structure of pAV.Des2d and pAV.Des3d of the present invention When building, the final positive rate for obtaining pAV.Des2d and pAV.Des3d is only respectively 1%, 1.2%.It can be seen that the method for the present invention is significant The recombination fraction of specificity is improved, recombinant clone accuracy significantly improves.

Respective element designation and Chinese in the pAV.Des3d of the pAV.Des2d and Fig. 5 of 1 Fig. 4 of table

The operating procedure schematic diagram of the present embodiment construction method is as shown in Figure 6.

PAV.Des2d the and pAV.Des3d adenovirus vector constructed below to the present invention makees further effect detection.

One, to the effect detection of pAV.Des2d adenovirus vector

Method：CMV promoter gene order and EGFP green fluorescence protein gene sequence are subjected to BP reaction respectively, must be entered Door clone pUp-CMV and pDown-EGFP.PUp-CMV, pDown-EGFP and the present invention are prepared into skeleton carrier pAV.Des2d again Step LR reaction is carried out, expression vector pAV [Exp]-CMV can be obtained>EGFP, by the expression vector transformed competence colibacillus cell, The indexs such as Positive rate, the accuracy of positive colony, virus titer.

As a result：Only pass through a step LR using pAV.Des2d of the present invention to react, can be obtained the positive containing 2 target fragments Expression vector pAV [Exp]-CMV>EGFP, and after PCR is identified, gained positive rate accuracy is 100%.Positive expression is carried Body pAV [Exp]-CMV>EGFP transfects 293A cell, and obvious lesion spot is found after 7 days, successfully packs adenovirus, it can be seen that very Strong green fluorescence expresses (Fig. 7), and qPCR detection virus titer can reach 10¹²PFU/ml。

Two, to the effect detection of pAV.Des3d adenovirus vector

Method：IRES-mCherry red fluorescent protein gene order is subjected to BP reaction, obtains entry clones pTail- IRES-mCherry.PUp-CMV, pDown-EGFP and pTail-IRES-mCherry and the present invention in above-mentioned " one " are prepared into bone Frame carrier pAV.Des3d carries out step LR reaction, and expression vector pAV [Exp]-CMV can be obtained>EGFP:IRES:MCherry, By the expression vector transformed competence colibacillus cell, the indexs such as Positive rate, the accuracy of positive colony, virus titer.

As a result：Only pass through a step LR using pAV.Des3d of the present invention to react, can be obtained the positive containing 3 target fragments Expression vector pAV [Exp]-CMV>EGFP:IRES:MCherry, and after PCR is identified, gained positive rate accuracy is 100%. By positive expression carrier pAV [Exp]-CMV>EGFP:IRES:MCherry transfects 293A cell, and obvious lesion spot is found after 7 days, Adenovirus is packed in success, it can be seen that very strong green and red fluorescence expression (Fig. 8), and qPCR detection virus titer highest can Reach 10¹²PFU/ml。

The above results explanation, the present invention are successfully realized the transformation to pAV.Des1d carrier, the pAV.Des2d constructed 2 or 3 mesh segments can be cloned simultaneously respectively with pAV.Des3d adenovirus vector, and it is anti-that common some segments are carried out BP Corresponding entry clones should be built into, then carry out free collocation according to demand, it is only necessary to which carrying out step LR reaction can be obtained Required adenovirus expression carrier, and there is good accuracy, high efficiency, success rate is high.Greatly reduce human time Cost improves user experience.The recombinant adenoviral vector that the present invention constructs can be arbitrarily introduced into as needed purpose biplate section or Three segments, it is more flexible quickly and efficiently to clone target fragment, improve production efficiency.

In addition, find in our current research, traditional Red/ET homologous recombination technique, can greatly increase second step recombination (on State the recombining reaction in 1 step 7 of embodiment) in occur unexpected intramolecular rearrangement, dystopy probability, there is false positive back Scape reduces positive rate.The abnormal conditions occurred in our Comprehensive Experiments delete the Red α gene on Red/ET carrier, excellent Change Red β and Red gamma gene sequences, is built into new carrier pRed/ET (Δ α).It is found in comparison reconstruction experiment, with original PRed/ET carrier is utilized to compare, the recombining reaction mediated using new pRed/ET (Δ α), although the colony counts on plate Reduce, but its positive rate has been increased to about 50% from original 1%, the accuracy of positive colony drops significantly close to 100% The low generation of false positive clones.This also solves us and endures second step recombination positive rate (1 step of above-described embodiment to the fullest extent all the time Recombining reaction in rapid 7) extremely low puzzlement, it improves work efficiency.

The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention, It should be equivalent substitute mode, be included within the scope of the present invention.

SEQUENCE LISTING

<110>Cloud boat biotechnology（Guangzhou）Co., Ltd

<120>A kind of adenovirus vector and its construction method

<130>

<160> 7

<170> PatentIn version 3.5

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gcataaaaaa cagactacat aatactgtaa aacacaacat atccagtcac tatggcggcc 180

gcattaggca ccccaggctt tacactttat gcttccggct cgtataatgt gtggattttg 240

agttaggatc cgtcgagatt ttcaggagct aaggaagcta aaatggagaa aaaaatcact 300

ggatatacca ccgttgatat atcccaatgg catcgtaaag aacattttga ggcatttcag 360

tcagttgctc aatgtaccta taaccagacc gttcagctgg atattacggc ctttttaaag 420

accgtaaaga aaaataagca caagttttat ccggccttta ttcacattct tgcccgcctg 480

atgaatgctc atccggaact ccgtatggca atgaaagacg gtgagctggt gatatgggat 540

agtgttcacc cttgttacac cgttttccat gagcaaactg aaacgttttc atcgctctgg 600

agtgaatacc acgacgattt ccggcagttt ctacacatat attcgcaaga tgtggcgtgt 660

tacggtgaaa acctggccta tttccctaaa gggtttattg agaatatgtt tttcgtctca 720

gccaatccct gggtgagttt caccagtttt gatttaaacg tggccaatat ggacaacttc 780

ttcgcccccg ttttcaccat gggcaaatat tatacgcaag gcgacaaggt gctgatgccg 840

ctggcgattc aggttcatca tgccgtttgt gatggcttcc atgtcggcag aatgcttaat 900

gaattacaac agtactgcga tgagtggcag ggcggggcgt aaacgcgtgg atccggctta 960

ctaaaagcca gataacagta tgcgtatttg cgcgctgatt tttgcggtat aagaatatat 1020

actgatatgt atacccgaag tatgtcaaaa agaggtatgc tatgaagcag cgtattacag 1080

tgacagttga cagcgacagc tatcagttgc tcaaggcata tatgatgtca atatctccgg 1140

tctggtaagc acaaccatgc agaatgaagc ccgtcgtctg cgtgccgaac gctggaaagc 1200

ggaaaatcag gaagggatgg ctgaggtcgc ccggtttatt gaaatgaacg gctcttttgc 1260

tgacgagaac aggggctggt gaaatgcagt ttaaggttta cacctataaa agagagagcc 1320

gttatcgtct gtttgtggat gtacagagtg atattattga cacgcccggg cgacggatgg 1380

tgatccccct ggccagtgca cgtctgctgt cagataaagt ctcccgtgaa ctttacccgg 1440

tggtgcatat cggggatgaa agctggcgca tgatgaccac cgatatggcc agtgtgccgg 1500

tctccgttat cggggaagaa gtggctgatc tcagccaccg cgaaaatgac atcaaaaacg 1560

ccattaacct gatgttctgg ggaatataaa tgtcaggctc ccttatacac agccagtctg 1620

caggtcgagc agatctgctc gaccatagtg actggatatg ttgtgtttta cagtattatg 1680

tagtctgttt tttatgcaaa atctaattta atatattgat atttatatca ttttacgttt 1740

ctcgttcagc tttcttgtac aaagtgggat cgattcgaca gatcactgaa atgtgtgggc 1800

gtggcttaag ggtggga 1817

<210> 3

<211> 1802

<212> DNA

<213>Artificial sequence

<400> 3

cttggatccg gtacctctag aattctcgag cggccgctag cgacatcgat caactttgta 60

tagaaaagtt gaacgagaaa cgtaaaatga tataaatatc aatatattaa attagatttt 120

gcataaaaaa cagactacat aatactgtaa aacacaacat atccagtcac tatggtcgac 180

ctgcagactg gctgtgtata agggagcctg acatttatat tccccagaac atcaggttaa 240

tggcgttttt gatgtcattt tcgcggtggc tgagatcagc cacttcttcc ccgataacgg 300

agaccggcac actggccata tcggtggtca tcatgcgcca gctttcatcc ccgatatgca 360

ccaccgggta aagttcacgg gggactttat ctgacagcag acgtgcactg gccaggggga 420

tcaccatccg tcgcccgggc gtgtcaataa tatcactctg tacatccaca aacagacgat 480

aacggctctc tcttttatag gtgtaaacct taaactgcat ttcaccagcc cctgttctcg 540

tcggcaaaag agccgttcat ttcaataaac cgggcgacct cagctatccc ttcctgattt 600

tccgctttcc agcgttcggc acgcagacga cgggcttcat tctgcatggt tgtgcttacc 660

gaaccggaga tattgacatc atatatgcct tgagcaactg atagctgtcg ctgtcaactg 720

tcactgtaat acgctgcttc atagcatacc tctttttgac atacttcggg tatacatatc 780

agtatatatt cttataccgc aaaaatcagc gcgcaaatac gcatactgtt atctggcttt 840

tagtaagccg gatcctctag attacgcccc gcctgccact catcgcagta ctgttgtaat 900

tcattaagca ttctgccgac atggaagcca tcacaaacgg catgatgaac ctgaatcgcc 960

agcggcatca gcaccttgtc gccttgcgta taatatttgc ccatggtgaa aacgggggcg 1020

aagaagttgt ccatattggc cacgtttaaa tcaaaactgg tgaaactcac ccagggattg 1080

gctgagacga aaaacatatt ctcaataaac cctttaggga aataggccag gttttcaccg 1140

taacacgcca catcttgcga atatatgtgt agaaactgcc ggaaatcgtc gtggtattca 1200

ctccagagcg atgaaaacgt ttcagtttgc tcatggaaaa cggtgtaaca agggtgaaca 1260

ctatcccata tcaccagccc accgtctttc attgccatac ggaattccgg atgagcattc 1320

atcaggcggg caagaatgtg aataaaggcc ggataaaact tgtgcttatt tttctttacg 1380

gtctttaaaa aggccgtaat atccagctga acggtctggt tataggtaca ttgagcaact 1440

gactgaaatg cctcaaaatg ttctttacga tgccattggg atatatcaac ggtggtatat 1500

ccagtgattt ttttctccat tttagcttcc ttagctcctg aaaatctcga cggatcctaa 1560

ctcaaaatcc acacattata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat 1620

gcggccgcca tagtgactgg atatgttgtg ttttacagta ttatgtagtc tgttttttat 1680

gcaaaatcta atttaatata ttgatattta tatcatttta cgtttctcgt tcaactttat 1740

tatacatagt tggatcgatt cgacagatca ctgaaatgtg tgggcgtggc ttaagggtgg 1800

ga 1802

<210> 4

<211> 1524

<212> DNA

<213>Artificial sequence

<400> 4

aaaccaattg tccatattgc atcagacatt gccgtcactg cgtcttttac tggctcttct 60

cgctaaccaa accggtaacc ccgcttatta aaagcattct gtaacaaagc gggaccaaag 120

ccatgacaaa aacgcgtaac aaaagtgtct ataatcacgg cagaaaagtc cacattgatt 180

atttgcacgg cgtcacactt tgctatgcca tagcattttt atccataaga ttagcggatc 240

ctacctgacg ctttttatcg caactctcta ctgtttctcc atacccgttt ttttgggcta 300

gcaggaggaa ttcaccatgg atattaacac cgaaaccgaa attaaacaga aacatagcct 360

gaccccgttt ccggtgtttc tgattagccc ggcgtttcgc ggccgctatt ttcatagcta 420

ttttcgcagc agcgcgatga acgcgtatta tattcaggat cgcctggaag cgcagagctg 480

ggcgcgccat tatcagcagc tggcgcgcga agaaaaagaa gcggaactgg cggatgatat 540

ggaaaaaggc ctgccgcagc atctgtttga aagcctgtgc attgatcatc tgcagcgcca 600

tggcgcgagc aaaaaaagca ttacccgcgc gtttgatgat gatgtggaat ttcaggaacg 660

catggcggaa catattcgct atatggtgga aaccattgcg catcatcagg tggatattga 720

tagcgaagtg taaaacgaat gagcaccgcg ctggcgaccc tggcgggcaa actggcggaa 780

cgcgtgggca tggatagcgt ggatccgcag gaactgatta ccaccctgcg ccagaccgcg 840

tttaaaggcg atgcgagcga tgcgcagttt attgcgctgc tgattgtggc gaaccagtat 900

ggcctgaacc cgtggaccaa agaaatttat gcgtttccgg ataaacagaa cggcattgtg 960

ccggtggtgg gcgtggatgg ctggagccgc attattaacg aaaaccagca gtttgatggc 1020

atggattttg aacaggataa cgaaagctgc acctgccgca tttatcgcaa agatcgcaac 1080

catccgattt gcgtgaccga atggatggat gaatgccgcc gcgaaccgtt taaaacccgc 1140

gaaggccgcg aaattaccgg cccgtggcag agccatccga aacgcatgct gcgccataaa 1200

gcgatgattc agtgcgcgcg cctggcgttt ggctttgcgg gcatttatga taaagatgaa 1260

gcggaacgca ttgtggaaaa caccgcgtat accgcggaac gccagccgga acgcgatatt 1320

accccggtga acgatgaaac catgcaggaa attaacaccc tgctgattgc gctggataaa 1380

acctgggatg atgatctgct gccgctgtgc agccagattt ttcgccgcga tattcgcgcg 1440

agcagcgaac tgacccaggc ggaagcggtg aaagcgctgg gctttctgaa acagaaagcg 1500

gcggaacaga aagtggcggc gtaa 1524

<210> 5

<211> 273

<212> DNA

<213>Artificial sequence

<400> 5

aaaccaattg tccatattgc atcagacatt gccgtcactg cgtcttttac tggctcttct 60

cgctaaccaa accggtaacc ccgcttatta aaagcattct gtaacaaagc gggaccaaag 120

ccatgacaaa aacgcgtaac aaaagtgtct ataatcacgg cagaaaagtc cacattgatt 180

atttgcacgg cgtcacactt tgctatgcca tagcattttt atccataaga ttagcggatc 240

ctacctgacg ctttttatcg caactctcta ctg 273

<210> 6

<211> 417

<212> DNA

<213>Artificial sequence

<400> 6

atggatatta acaccgaaac cgaaattaaa cagaaacata gcctgacccc gtttccggtg 60

tttctgatta gcccggcgtt tcgcggccgc tattttcata gctattttcg cagcagcgcg 120

atgaacgcgt attatattca ggatcgcctg gaagcgcaga gctgggcgcg ccattatcag 180

cagctggcgc gcgaagaaaa agaagcggaa ctggcggatg atatggaaaa aggcctgccg 240

cagcatctgt ttgaaagcct gtgcattgat catctgcagc gccatggcgc gagcaaaaaa 300

agcattaccc gcgcgtttga tgatgatgtg gaatttcagg aacgcatggc ggaacatatt 360

cgctatatgg tggaaaccat tgcgcatcat caggtggata ttgatagcga agtgtaa 417

<210> 7

<211> 786

<212> DNA

<213>Artificial sequence

<400> 7

atgagcaccg cgctggcgac cctggcgggc aaactggcgg aacgcgtggg catggatagc 60

gtggatccgc aggaactgat taccaccctg cgccagaccg cgtttaaagg cgatgcgagc 120

gatgcgcagt ttattgcgct gctgattgtg gcgaaccagt atggcctgaa cccgtggacc 180

aaagaaattt atgcgtttcc ggataaacag aacggcattg tgccggtggt gggcgtggat 240

ggctggagcc gcattattaa cgaaaaccag cagtttgatg gcatggattt tgaacaggat 300

aacgaaagct gcacctgccg catttatcgc aaagatcgca accatccgat ttgcgtgacc 360

gaatggatgg atgaatgccg ccgcgaaccg tttaaaaccc gcgaaggccg cgaaattacc 420

ggcccgtggc agagccatcc gaaacgcatg ctgcgccata aagcgatgat tcagtgcgcg 480

cgcctggcgt ttggctttgc gggcatttat gataaagatg aagcggaacg cattgtggaa 540

aacaccgcgt ataccgcgga acgccagccg gaacgcgata ttaccccggt gaacgatgaa 600

accatgcagg aaattaacac cctgctgatt gcgctggata aaacctggga tgatgatctg 660

ctgccgctgt gcagccagat ttttcgccgc gatattcgcg cgagcagcga actgacccag 720

gcggaagcgg tgaaagcgct gggctttctg aaacagaaag cggcggaaca gaaagtggcg 780

gcgtaa 786

Claims

1. a kind of construction method of adenovirus vector, which is characterized in that include the following steps：

3) product after recovery culture is applied to the culture simultaneously containing ammonia benzyl antibiotic Amp, chloramphenicol Cm and tetracycline Tc On base plate, it is protected from light culture, picking positive colony bacterium A；

4) inducing expression homologous recombination enzyme in the culture medium containing L-arabinose is added in positive colony bacterium A obtained by upper step and prepared At competent cell, Kan resistance expression's box PCR product with homology arm is transferred to and wherein carries out recombining reaction, recovery culture；

5) the recovery cultured products of step 4) are applied on the culture medium flat plate containing Amp, Kan and Tc, are protected from light culture, picking weight Group positive colony bacterium B；

6) the difference liquid medium within of positive colony bacterium B obtained in step 5) is expanded into culture, wherein containing Amp, Kan and Tc Resistance culture base in the clone bacterium that grows, and cannot be grown in the base of resistance culture containing Cm be purpose positive colony bacterium C；

7) L-arabinose inducing expression homologous recombination enzyme is added in purpose positive colony bacterium C obtained in step 6) and be prepared into Competent cell, by the attR4-Cm-ccdB-attR2 with homology arm or the attR4-Cm-ccdB-attR3 segment with homology arm It is transferred to and wherein carries out recombining reaction；Recovery culture；

8) recovery cultured products in step 7) are applied on the culture medium flat plate containing Amp, Cm and Tc, 36.5~37.5 DEG C of trainings It supports, picking recombinant clone bacterium D；

9) the difference liquid medium within of positive colony bacterium D obtained in step 8) is expanded into culture, wherein anti-containing Amp and Cm The clone bacterium that grows in property culture medium, and cannot grow in containing Kan and Tc resistance culture base is purpose positive colony bacterium E； The cultivation temperature of all cultures is 36.5~37.5 DEG C in the step；

2. the method according to claim 1, wherein in step 1), also to Red γ in pRed/ET (Δ α) carrier It is optimized with Red beta gene sequence, Red γ and the Red beta gene sequence after optimization are respectively such as SEQ ID NO:6,SEQ ID NO:Shown in 7.

3. the method according to claim 1, wherein step 1)~7) in the cultivation temperatures of all cultures be 29.5 ~30.5 DEG C.

4. the method according to claim 1, wherein the base sequence of the Kan resistance fragments with homology arm Such as SEQ ID NO:Shown in 1.

5. the method according to claim 1, wherein the attR4-Cm-ccdB-attR2 with homology arm Base sequence such as SEQ ID NO:Shown in 2.

6. the method according to claim 1, wherein the attR4-Cm-ccdB-attR3 with homology arm Base sequence such as SEQ ID NO:Shown in 3.

7. the adenovirus vector of any the method building of claim 1~6.

8. the thallus containing adenovirus vector described in claim 7.

9. a kind of adenovirus vector pAV.Des2d, which is characterized in that compared with existing pAV.Des1d carrier, distinctive points exist for it In, pAV.Des2d with attR4 instead of the attR1 sequence in pAV.Des1d, while pAV.Des2d is connected in the downstream attR2 Terminator sequence.

10. a kind of adenovirus vector pAV.Des3d, which is characterized in that compared with existing pAV.Des1d carrier, distinctive points exist for it In, pAV.Des3d with attR4 instead of the attR1 sequence in pAV.Des1d, while with attR3 instead of in pAV.Des1d AttR2 sequence, and pAV.Des3d is connected with terminator sequence in the downstream attR3.