CN103290052B - A kind of adenovirus system of improvement and the Synthesis and applications of virion thereof - Google Patents

Abstract

The invention discloses an improved adenovirus vector system and a virus preparation method. The pDONR221 vector is used as the initial vector for improvement, and multiple cloning sites and IRES-GFP fragments are inserted. The system combines restriction enzyme ligation and recombination reaction technology, which can not only efficiently construct the vector, but also reduce the cost of reagents. The invention also discloses an improved purification method after enzymatic cleavage of the subsequent viral vector, which abandons the traditional phenol: chloroform: isoamyl alcohol purification method, and uses a conventional DNA recovery column or direct heating inactivation method, which is simple and easy , shorten the operation time, reduce the cost of materials, and do not need to use toxic phenol and chloroform reagents. Using the adenovirus system combined with these two aspects, the preparation efficiency is improved, the cost is reduced, and it still maintains high efficiency in infecting cells.

Description

Preparation and application of an improved adenovirus vector system and its virus particles

technical field

The invention belongs to the field of biotechnology, in particular to an improved adenovirus vector system and the preparation and application of virus particles.

Background technique

The development of adenovirus (adenovirus, Ad) as a gene expression carrier began in the early 1960s, when virologists observed that the adenovirus genome could hybridize with the simian virus 40 (SV40) genome, indicating that the adenovirus genome can carry heterogeneous source gene. Since then, adenovirus has gradually developed into an important vector system.

Adenovirus vectors are characterized by high transgene efficiency, in vitro experiments usually have a transduction efficiency of more than 90%; they can transduce different types of human tissue cells, regardless of whether the target cells are dividing cells; they do not integrate into cells The host cell genome is only expressed transiently, with high safety. Therefore, adenoviral vectors have been used more and more in clinical trials of gene therapy, and have become the most widely used and most promising viral vectors after retroviral vectors. The most widely used adenovirus vector is serotype 5 adenovirus.

At present, there are two main strategies for constructing recombinant adenovirus system: recombination in cells/bacteria and direct recombination in vitro. The former is represented by Stratagene’s Ad-easy system. The recombination reaction is carried out in cells or bacteria, and cumbersome screening steps are required to obtain the correct recombinant vector; the latter uses in vitro recombination method, based on Clontech’s Adeno-X system and Invitrogen The company's Virapower system is represented. The Adeno-X system uses two enzyme-cut ligation methods, but the success rate is low due to the long viral genome (greater than 36K) in the second ligation, and the kit is expensive. Invitrogen's Virapower system uses two recombination methods to complete the recombination vector in vitro, but the two recombination requires two recombinases, which is very expensive, and the recombination enzyme is sensitive to temperature, which often leads to unstable recombination efficiency.

The vectors completed in vitro recombination generally need to be digested with PacI before packaging the virus, and purified by the classic phenol: chloroform: isoamyl alcohol. This method takes a long time, the recovery efficiency is not high, and there is a risk of chemical hazards to the operator.

In order to make the adenovirus system 1) achieve high efficiency, reduce instability, and reduce reagent costs during the vector construction process; 2) improve the purification recovery rate and avoid toxic chemical With the use of reagents, we have improved the adenovirus vector system and the purification method after enzyme digestion, which has improved efficiency, reduced costs, and achieved good virus particle preparation results.

Contents of the invention

The purpose of the present invention is to provide an improved adenovirus vector system and virus preparation method, the system combines enzyme cleavage ligation and recombination reaction technology, which can efficiently construct the vector and reduce the cost of reagents; another purpose of the present invention is to provide An improved method of purification after enzymatic digestion of subsequent viral vectors. This method abandons the traditional phenol: chloroform: isoamyl alcohol method, and uses conventional DNA recovery columns or direct heat inactivation methods, which are simple and easy to implement and improve efficiency. , using the adenovirus system combining these two aspects, the preparation efficiency is improved and the cost is reduced.

Technical scheme of the present invention is as follows:

The purpose of the present invention is to provide an improved adenovirus vector system and virus preparation method, the system combines enzyme cleavage ligation and recombination reaction technology, which can efficiently construct the vector and reduce the cost of reagents; another purpose of the present invention is to provide An improved method of purification after enzymatic digestion of subsequent viral vectors. This method abandons the traditional phenol: chloroform: isoamyl alcohol method, and uses conventional DNA recovery columns or direct heat inactivation methods, which are simple and easy to implement and improve efficiency. , using the adenovirus system combining these two aspects, the preparation efficiency is improved and the cost is reduced.

Technical scheme of the present invention is as follows:

One of the technical schemes of the present invention is: an adenovirus vector system, which is improved by using pDONR221 vector as the initial vector, and inserting multiple cloning sites and IRES-GFP fragments.

Wherein, preferably, the multiple cloning sites are: NotI, XbaI, BglII, XhoI, SacI, EcoRI, PstI, SalI, SacII, SmaI, BamHI in sequence.

The present invention starts with the backbone part of Invitrogen's vector pDONR221, and connects multiple cloning sites and IRES-GFP fragments, so that the vector can highly express the target gene, and at the same time, GFP protein is used as a tracer, which overcomes the lack of GFP in the original vector Defects in the tracer protein.

When designing multiple cloning sites, other sequence information on the vector was fully evaluated, plus a single restriction site: NotI, XbaI, BglII, XhoI, SacI, EcoRI, PstI, SalI, SacII, SmaI, BamHI, which are beneficial Cloning is carried out by simple digestion and ligation. The sequence information of the restriction site is as follows:

5'-GCGGCCGCTTCTAGACTCAGATCTCGAGCTCAAGCTTCGAATTCTGCAGTCGACGGTACCGCGGGCCCGGGATCC-3'.

Wherein, preferably, the adenovirus vector is prepared by a method comprising the following steps:

1) Take the pIRES-EGFP vector as a template, and use the following two primers for PCR amplification to obtain the MCS-IRES-GFP fragment. Among the primers, the nucleotide sequence of one primer is shown in SEQ ID NO.1, and the other The nucleotide sequence of the primer is shown in SEQ ID NO.2;

2) BP recombination of the above MCS-IRES-GFP fragment and the vector pDONR221 to obtain the vector pDONR-MCS-IRES-GFP;

3) Perform LR recombination of the vector pDONR-MCS-IRES-GFP and the vector pAd/CMV/V5-DEST to obtain the final vector pAd/MCS-IRES-GFP.

Wherein, the BP recombination is conventional in the art, and refers to the specific recombination of the attB site and the attP site. The BP recombination is preferably carried out using Invitrogen's BP recombination system. The LR recombination is conventional in the art, and refers to specific recombination attL and attR sites. The LP recombination preferably uses the LR recombination system of Invitrogen for LR recombination.

The second technical solution of the present invention is: a recombinant adenoviral vector, which is a recombinant adenoviral vector in which foreign gene fragments are inserted into the multi-cloning site of the adenoviral vector.

The third technical solution of the present invention is: a method for packaging viruses with the recombinant adenoviral vector, comprising transfecting 293A cells after linearizing the recombinant adenoviral vector, including:

1) Digesting the recombinant adenovirus vector with PacI restriction endonuclease;

2) Purify the enzyme digestion reaction solution obtained in step 1) and recover the enzyme digestion product for transfection; or heat up the enzyme digestion reaction solution obtained in step 1) to inactivate it, and then directly apply it to transfection.

Preferably, in step 1), the amount of the recombinant adenovirus vector is 0.01-1 μg/μl, and the amount of PacI enzyme is 0.01-1 U/μl; more preferably, the enzyme-cut amount of the adenovirus vector is 5-20μg, the dosage of PacI enzyme is 5-20U, and the enzyme digestion reaction volume is 20μl-100μl.

In step 2), the purification refers to the purification of conventional enzyme digestion products, and conventional column purification methods can be used.

In step 2), the heating inactivation is preferably at a temperature of 65° C. and a holding time of 20 minutes.

The present invention adopts the lipofectamine2000 method on the adenovirus particle preparation method, and the specific steps that a preferred example comprises are as follows

1) Take 293A cells in the logarithmic growth phase, digest with 0.05% trypsin, count the cells, plant them in a 6-well plate at 4.5×10 ⁵ cells per well, and culture overnight;

2) Remove the culture medium before transfection the next day, and replace with 2ml of fresh culture medium;

3) Add 2 μg of enzyme-digested plasmid to 250 μl of Opti-MEM preheated at 37°C, and mix well;

4) Add 5 μl lipofectamine2000 to 250 μl Opti-MEM, mix well, and place at room temperature for 5 minutes;

5) Mix the purified digested plasmid or inactivated plasmid solution and diluted lipofectamine2000, and place at room temperature for 20 minutes;

6) Add 500μl of the complex to the cell well and mix well. After culturing in an incubator with 5% CO2 at 37°C for 5-6 hours, replace with complete culture medium and continue culturing in the incubator;

7) 48 hours after transfection, the digested cells were transferred to a 10cm culture dish, and 10ml of complete culture medium was added;

8) Change into fresh medium every 2-3 days, and observe whether there are lesions (CPE) (usually 7-10 days after transfection);

9) Allow the infection to continue until 80% CPE appears (usually 10-13 days after transfection). Collect cells and supernatants into sterile 15ml centrifuge tubes. Collect cells and supernatants into sterile 15ml centrifuge tubes. Freeze and thaw repeatedly 3 times, centrifuge at 3000g for 10 minutes, collect the supernatant, and store it in a -70°C refrigerator.

The adenoviral particle of the invention can be used for gene transduction of cells and transduction in animals, has simple operation, high transduction efficiency, and can effectively infect various cells.

The adenoviral vector of the present invention carries a marker green fluorescent protein (GFP) and a multiple cloning site.

The raw materials or reagents used in the present invention are commercially available unless otherwise specified.

Compared with the prior art, the beneficial effects of the present invention are as follows:

1. In the present invention, pDONR221 has been improved, and multiple cloning sites have been newly added, so that the new vector can be constructed by simple enzyme cutting and ligation methods, eliminating the BP recombination reaction, and only need to carry out one more LR recombination reaction The final target carrier can be constructed, the whole process is more efficient, and the reagent cost is reduced by nearly half.

2. The present invention changes the method of using phenol: chloroform: isoamyl alcohol in the purification process after the carrier digestion, and adopts the conventional PCR purification kit, or adopts the direct temperature rise inactivation method, so that the operation time is greatly shortened, Moreover, the recovery efficiency is improved, and the use of harmful chemical reagents can be avoided.

Description of drawings

The features and beneficial effects of the present invention will be described below in conjunction with the accompanying drawings.

Figure 1 is the original vector map of pDONR221.

Fig. 2 is a map of the transformed vector pDONR-MCS-IRES-GFP.

Figure 3 is a map of the vector pAd/CMV/V5-DEST.

Fig. 4 is a schematic diagram of vector construction in Examples 1 and 2.

Fig. 5 is a schematic diagram of vector construction in Example 3.

Fig. 6 is a schematic diagram of vector construction in Example 6 and Example 7.

Figure 7 shows the comparison of virus titers obtained by different treatment methods after digestion of the empty vector adenovirus. 1. Phenol-chloroform method; 2. Column purification method; 3. Heat denaturation method.

Figure 8 shows the comparison of virus titers obtained by different treatment methods after digestion of the adenoviral vector carrying the LacZ exogenous gene. 1. Phenol-chloroform method; 2. Column purification method; 3. Heat denaturation method.

detailed description

The present invention is further illustrated below with examples, but the present invention is not limited thereto. For the experimental methods without specific conditions indicated in the following examples, the conventional conditions or the conditions suggested by the manufacturer are usually followed. The "room temperature" mentioned in the examples refers to the temperature in the operating room where the test is carried out, which is generally 25°C.

Example 1

The amplified MCS-IRES-GFP fragment is as follows:

Design and synthesize 2 PCR primers, the sequences are as follows:

5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTGCGGCCGCTTTCTAGACTCAGATCTCGAGCT-3' (SEQ ID NO. 1);

5' - GGGGACCACTTTGTACAAGAAAGCTGGGTCTTACTTGTACAGCTCGTCCAT-3' (SEQ ID NO. 2).

The pIRES-EGFP vector (purchased from Clontech) was used as a template, and the above two oligonucleotide chains were used as primers to amplify to obtain the DNA fragment of MCS-IRES-GFP. The fragment amplified from the pIRES-EGFP vector contains multiple cloning sites NotI, XbaI, BglII, XhoI, SacI, EcoRI, PstI, SalI, SacII, SmaI, and BamHI.

1) The volume of each component in the PCR system

Component volume Ultra-pure water 37.8μl 10XPCR buffer 5μl Magnesium ion (25mM) 3μl dNTPs (25mM) 0.4μl Upstream primer (10μM) 1μl Downstream primer (10μM) 1μl Taq enzyme (5U/μl) 0.3μl DNA template 1.5μl overall system 50μl

PCR reaction conditions:

①: 95℃, 2 minutes;

② (30 cycles): 95°C, 30 seconds, 55°C, 30 seconds, 72°C, 60 seconds;

③: 72°C, 10 minutes;

2) PCR products were purified with AxygenPCR Clean Kit.

Example 2

The pDONR221 vector (Invitrogen, Figure 1) was transformed into a pDONR-MCS-IRES-GFP vector, including purification of the amplified MCS-IRES-GFP fragment, and BP recombination with pDONR221, and the obtained vector was named pDONR-MCS-IRES -GFP (see Figure 2). details as follows:

Use Invitrogen's BP recombination system (Invitrogen's BP recombination kit) to recombine the target fragment into the vector pDONR221. Prepare the BP recombination reaction system according to the following table:

PCR product 2μl pDONR221 plasmid 1μl BP clonaseⅡenzyme mix 2μl TE buffer (pH8.0) 5μl

React at 25°C for 1 hour.

Add 1 μl of proteinase K and react at 37°C for 10 minutes.

Take 5 μl of the recombination reaction solution to transform 50 μl of Escherichia coli DH5α competent cells.

Positive clones were screened and verified by sequencing, and the correct BP recombinant plasmids verified by sequencing were retained.

See Figure 4 for a schematic diagram of the vector construction process.

Example 3

The pDONR-MCS-IRES-GFP vector prepared in Example 2 and the pAd/CMV/V5-DEST vector (purchased from Invitrogen, Figure 3) were subjected to LR recombination to obtain the final recombinant vector pAd/MCS-IRES-GFP. details as follows:

LR recombination was performed using Invitrogen's LR recombination system (Invitrogen's LR recombination kit). Prepare the LR recombination reaction system according to the following table:

BP recombinant plasmid 1μl pAd CMV/V5-DEST 1μl LR clonaseⅡenzyme mix 2μl TE buffer (pH8.0) 6μl

React at 25°C for 1 hour.

Add 1 μl of proteinase K and react at 37°C for 10 minutes.

Take 5 μl of the recombination reaction solution to transform 50 μl of DH5α competent cells.

Positive clones were screened and verified by sequencing, and the correct LR recombinant plasmids verified by sequencing were retained.

See Figure 5 for a schematic diagram of the vector construction process.

Example 4

Enzyme digestion reaction: PacI digestion vector pAd/MCS-IRES-GFP, the reaction system is 1× digestion buffer, 5-20μg recombinant vector, 5-20UPacI enzyme, the total volume is 20-100μl.

Recovery of digested products: Use a commonly used PCR cleaning kit (purchased from Axygen), add buffer to the digested product according to the instruction manual at 3 times the volume, mix well, add to the purification column, centrifuge at 10000g for 1 minute, and add washing buffer 600μl, twice, centrifuged at 10000g for 1 minute, then centrifuged at 12000g for 1 minute to remove the residual liquid, and added 30μl of water to elute the DNA solution. DNA concentration was quantified by UV spectrophotometer.

Alternatively, incubate the solution of the restriction enzyme digestion reaction at 65°C for 20 minutes, and directly transfect the required amount of DNA into 293A cells.

Example 5

Adenovirus packaging:

1) Take 293A cells in good condition and in the logarithmic growth phase, digest with 0.05% trypsin, count the cells, plant them in a 6-well plate at 4.5×10 ⁵ cells per well, at 37°C, 5% CO ₂ overnight in an incubator;

2) Remove the culture medium before transfection the next day, and replace with 2ml of fresh culture medium;

3) Add 2 μg of the aforementioned enzyme-digested plasmid (recovered product obtained in Example 4, or enzyme digestion reaction solution) into 250 μl of Opti-MEM (purchased from Invitrogen) preheated at 37°C, and mix well;

4) Add 5μllipofectamine2000 (purchased from Invitrogen) into 250ul Opti-MEM, mix well, and place at room temperature for 5min;

5) Gently mix the purified digested plasmid or inactivated plasmid solution and diluted lipofectamine2000, and place at room temperature for 20 minutes;

6) Add 500 μl of the complex to the cell well, shake the culture plate, and mix well. After culturing in an incubator with 5% CO2 at 37°C for 5-6 hours, replace with complete culture medium and continue culturing in the incubator;

7) 48 hours after transfection, the digested cells were transferred to a 10cm culture dish, and 10ml of complete medium was added;

8) Change into fresh medium every 2-3 days, and observe whether there are lesions (CPE) (usually 7-10 days after transfection);

9) Allow the infection to continue until 80% CPE appears (usually 10-13 days after transfection). Collect cells and supernatants into sterile 15ml centrifuge tubes. Freeze and thaw repeatedly 3 times, centrifuge at 3000g for 10 minutes, collect the supernatant, and store it in a -70°C refrigerator, which is the adenovirus solution. It has been determined that the titer of the virus packaged by the vector obtained by the column purification method is about 1E9pfu/ml, while the virus titer of the packaged virus obtained by the vector denatured by heating and omitting the purification step is about 4E8pfu/ml (for titer determination, refer to the classic Cell spot method, refer to Invitrogen instructions).

The titer results of viruses packaged using other conventional methods are shown in FIG. 7 . Compared with the classic phenol-chloroform purification method (see Invitrogen product manual), the titer of the virus prepared by the column purification method is higher; also compared with the simplified thermal denaturation method of the present invention, the virus can still be successfully packaged, and the titer does not change much , but compared with the conventional phenol-chloroform purification method, the whole step saves time, avoids the use of toxic chemical reagents, is conducive to environmental protection, and saves costs.

Example 6

Using Escherichia coli LacZ gene as an exogenous gene, test the vector pDONR-MCS-IRES-GFP of the present invention, first amplify LacZ by PCR method, and clone it into pDONR-MCS-IRES-GFP vector, and use the LacZ of the template The vector is pLenti6.3/V5/GW-LacZ (product of Invitrogen). See Figure 6 for a schematic diagram of the vector construction process.

Specific steps are as follows

1. PCR amplification of exogenous gene LacZ:

1) Design a pair of PCR primers to amplify the LacZ gene,

Upstream primer LacZ-F (with XhoI site):

5'-TTATTCTCGAGACCATGATAGATCCCGTCGTTTTACA-3';

Downstream primer LacZ-R (contains EcoRI site):

5'-TTATTGAATTCTTAGTAGTTTTTTGACACCAGACCAACTG-3'.

2) PCR reaction conditions:

The volume of each component in the PCR system:

Component volume Ultra-pure water 37.8μl 10XPCR buffer 5μl Magnesium ion (25mM) 3μl dNTPs (25mM) 0.4μl Upstream primer LacZ-F (10μM) 1μl Downstream primer LacZ-R (10μM) 1μl Taq enzyme (5U/μl) 0.3μl DNA template 1.5μl overall system 50μl

PCR reaction program:

①: 95℃, 2 minutes;

② (30 cycles): 95°C, 30 seconds, 55°C, 30 seconds, 72°C, 3 minutes;

③: 72°C, 10 minutes;

3) PCR products were purified with AxygenPCR cleaning kit;

4) Digest the fragment with XhoI and EcoRI enzymes. The enzyme digestion system is: 2 μl of 10-fold buffer, 2 μg of purified PCR product, 10 units each of XhoI and EcoRI enzymes, and the total volume is 20 μl;

5) The vector pDONR-MCS-IRES-GFP modified by this patent was digested with XhoI and EcoRI, and purified with AxygenPCR cleaning kit;

6) Ligation reaction: add 0.2 μg of digested and purified PCR product, 0.1 μg of vector after digestion, add 2 μl of 10×T4 ligase buffer, 1 μl of T4 ligase, total volume 20 μl, 16°C for 4 hours;

7) Transform Escherichia coli, pick a single colony, extract the plasmid, send it for sequencing verification, and select the vector with the correct sequence for LR recombination reaction.

Example 7

LR recombination constructs the final adenoviral vector containing LacZ gene and viral packaging.

1. Perform LR recombination of the pDONR-MCS-IRES-GFP vector inserted into the LacZ gene prepared in Example 6 and the pAd/CMV/V5-DEST vector (purchased from Invitrogen) to obtain the final recombinant vector pAd/LacZ-IRES-GFP, Concrete steps are with embodiment 3.

2. PacI digestion of pAd/LacZ-IRES-GFP vector and purification method, the specific steps are the same as in Example 4.

3. Adenovirus packaging: the specific steps are the same as in Example 5. The titer of the virus packaged by the column purification method is about 8.6E8pfu/ml, while the titer of the virus directly packaged by simple heat denaturation is 4E8pfu/ml, compared with the titer of the classic phenol: chloroform purification method is 4.5E8pfu/ml ( See Figure 8). For titer determination, refer to the classic cell spot method and refer to the instructions of Invitrogen.

Example 8

Adenovirus infection of target cells (target cells are Hela cells (purchased from Shanghai Cell Bank, Chinese Academy of Sciences):

1. The target cells are seeded in 96-well plates one day before virus infection, seeding plate volume: 2.0×10 ⁴ cells/well;

2. On the day of infection, replace the 96-well plate with freshly prepared medium: 50 μl/well, add the adenovirus solution (prepared in Examples 5 and 7) in the range of MOI=0-100, mix well and place in cell culture box;

3. About 6 hours after the addition of the virus, replace the fresh medium in the 96-well plate, and continue culturing in an incubator at 37°C and 5% CO ₂ . Observe the infection results for 48-96 hours. When the MOI=20 or more, the positive rate of cells infected by the virus is more than 90%, which reflects that the prepared adenovirus has good vigor and can be completely applied to gene transduction of cells.

It should be understood that after reading the above content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (4)

1. a method for recombinant adenoviral vector packaging virus, comprises transfection 293A cell after recombinant adenoviral vector linearizing, it is characterized in that, comprising:

1) recombinant adenoviral vector PacI restriction enzyme multiple clone site being inserted exogenous genetic fragment carries out endonuclease reaction, and wherein said recombinant adenoviral vector improves for starting vector with pDONR221 carrier, inserts multiple clone site and IRES-GFP fragment, described multiple clone site is followed successively by: NotI, XbaI, BglII, XhoI, SacI, EcoRI, PstI, SalI, SacII, SmaI, BamHI;

2) by step 1) the endonuclease reaction liquid purifying of gained reclaim digestion products and be applied to transfection; Or by step 1) the endonuclease reaction liquid intensification deactivation of gained, then directly apply to transfection, the method for wherein said endonuclease reaction liquid purifying is column purification method.

2. the method for claim 1, is characterized in that, step 1) in, the consumption of described recombinant adenoviral vector is 0.01-1 μ g/ μ l, and the consumption of PacI enzyme is 0.01-1U/ μ l; Step 2) described in intensification deactivation temperature 65 DEG C, soaking time 20 minutes.

3. the method for claim 1, is characterized in that, step 1) described in adenovirus carrier prepared by the method that comprises the following steps and obtain:

1) getting pIRES-EGFP carrier is template, adopt following two primers to carry out pcr amplification, obtain MCS-IRES-GFP fragment, in described primer, article one, the nucleotide sequence of primer is as shown in SEQIDNO.1, and the nucleotide sequence of another primer is as shown in SEQIDNO.2;

2) by step 1) described in MCS-IRES-GFP fragment and carrier pDONR221 carry out BP restructuring, obtain carrier pDONR-MCS-IRES-GFP;

3) carrier pDONR-MCS-IRES-GFP and carrier pAd/CMV/V5-DEST is carried out LR restructuring, obtain final carrier pAd/MCS-IRES-GFP.

4. method as claimed in claim 3, is characterized in that, step 2) described in BP restructuring use the BP recombination system of Invitrogen to carry out BP restructuring; Step 3) described in LR restructuring use the LR recombination system of Invitrogen to carry out LR restructuring.