WO2014026503A1

WO2014026503A1 - Method for targeted knockout of non-essential genes for bombyx mori nuclear polyhedrosis virus replication

Info

Publication number: WO2014026503A1
Application number: PCT/CN2013/077515
Authority: WO
Inventors: 田凤鸣; 陈剑清; 舒特俊; 张耀洲
Original assignee: 天津耀宇生物技术有限公司
Priority date: 2012-08-14
Filing date: 2013-06-20
Publication date: 2014-02-20
Also published as: CN103589745B; CN103589745A

Abstract

The present invention relates to a method for targeted knockout of non-essential genes for replication of the Bombyx mori nuclear polyhedrosis virus BmNPV comprising: using BmNPV as material to amplify a homologous sequence at both ends of a non-essential region for replication by PCR and cloning it into vector PUC19; splicing successively IE1 early promoter, marker gene (EGFP) and termination sequence SV40polyA of BmNPV by overlapping PCR and cloning them to the abovementioned vector pUC19 to obtain the recombinant transfer vector pUC19-lef7-IE1-EGFP-SV40polyA-gp64; co-transfecting BmN cells using the vector and the genome of wild BmNPV to obtain the recombinant virus RBmNPV-EGFP with a fluorescent marker via homologous recombination; co-transfecting BmN cells using genomic DNA thereof and transferring vector pUC19-lef7-gp64 without marker gene to obtain recombinant virus RBmNPV without fluorescent marker gene via homologous recombination. The present invention solves the problem of the presence of marker genes in recombinant virus genomes, improving the screening efficiency of positive recombinant viruses and thus the marker genes can be reused.

Description

FIELD OF THE INVENTION The present invention relates to the field of DNA recombination technology and gene targeting technology, and more particularly to a targeted knockout of a non-essential gene for the replication of the Bombyx mori nuclear polyhedrosis virus. Methods. Background technique

Gene knockout was developed using the principle of DNA homologous recombination in the second half of the 1980s. At the beginning of the 1980s, the success of embryonic stem cell (ES cell) isolation and in vitro culture laid the foundation for gene knockout. In 1985, the existence of homologous recombination in mammalian cells, first confirmed, laid the theoretical foundation for gene knockout. By 1987, Thompsson first established a complete mouse model of ES cell knockout. Until now, gene knockout using gene homologous recombination remains the most common method of constructing gene knockout animal models. It is also an important means of studying gene function. Gene knockout technology is now widely used in the study of viral genome function. Therefore, a replacement vector is generally designed to co-transfect cells with the viral genome to delete genes. However, the problem of leaving the marker gene on the viral genome has not been fully improved. This has had a huge impact on the function of the virus itself and the subsequent virus screening has caused great trouble. Therefore, we used it twice. The method of homologous recombination not only deletes the target gene but also solves the problem of the labeled gene on the viral genome, which is of great significance for future scientific experiments. Summary of the invention

In view of the above, one of the objects of the present invention is to provide a marker-bearing transfer vector pUC 19-IE 1 -EGFP-S V40polyA which can knock out the non-essential gene of Bombyx mori nuclear polyhedrosis virus BmNPV and preparation method thereof .

In order to achieve the above object, the present invention provides a preparation vector of a transfer vector carrying a marker gene. Method, the method includes the following steps:

1) The genes are sequentially obtained by PCR: IE, EGFP, SV40polyA;

2) IE1, EGFP were spliced by overlapping PCR method, and the ligated product of IE1-EGFP was spliced with the gene SV40polyA to obtain the triplet gene IEl-EGFP-SV40polyA;

3) The triplet gene IEl-EGFP-SV40 polyA obtained in the step 2) was cloned into the vector pUC19 to obtain the transfer vector UC 19-IE 1 -EGFP-S V40 polyA;

4) Transfer vector UC 19-IE 1 -EGFP-SV40 polyA transforms DH5a E. coli competent cells;

5) Blue-white spot screening (solid medium containing ampicillin), and white colonies containing the transfer vector pUC19-IEl-EGFP-SV40polyA were picked.

6) Transfer vector UC 19-IE 1 -EGFP-S V40 polyA was identified by PCR and double enzyme digestion and sent to the Huada gene for sequencing.

The present invention further provides a transfer vector carrying a marker gene, which is produced by the above method.

Another object of the present invention is to provide a method for knocking out a non-essential gene for the knockout of a Bombyx mori nuclear polyhedrosis virus to solve the problem of containing a marker gene on a recombinant virus genome, and the marker gene can be reused.

In order to achieve the above object, the present invention also provides a method for targeting a knockout silkworm nuclear polyhedrosis virus to replicate a non-essential gene, the method comprising the steps of:

1) Extracting the BmNPV genome of the Bombyx mori nuclear polyhedrosis virus;

2) PCR method to obtain homologous sequences LEF7, gp64 at both ends of the replication non-essential fragment;

3) The homologous sequences lef7 and gp64 obtained in the step 2) were cloned into the above-mentioned transfer vector pUC 19-IE 1 -EGFP-S V40polyA to obtain a recombinant transfer vector pUC 19-lef7-IE 1 -EGFP-S V40polyA-gp64 And with the silkworm nuclear polyhedrosis virus genome The BmN cells were transfected, and the recombinant virus RBmNPV-EGFP carrying the EGFP marker (fluorescent protein marker gene) was obtained by homologous recombination, and the recombinant virus RBmNPV-EGFP was identified and expanded.

4) Extraction of recombinant virus RBmNPV-EGFP genome and marker-free gene transfer vector pUC19-lef7-gp64 (by liposome-mediated transfection) co-transfection of BmN cells;

5) Screening of the marker-free recombinant virus RBmNPV, expanded culture for subsequent experiments. The beneficial effects of the invention: First, the transfer carrier constructed by the invention can be continuously knocked out

The non-essential genes of different transcriptional phases in the BmNPV genome do not need to construct too many vectors; the second is to use green fluorescent protein (EGFP) as a marker gene to obtain recombinant virus quickly and intuitively; The problem of the marker gene contained in the recombinant viral genome is solved, and the marker gene can be reused without replacing the marker gene. DRAWINGS

Figure 1 shows the electrophoresis patterns of three genes: IE1, EGFP, and SV40polyA; M: lOkb DNA marker; 1: PCR product of IE1; 2: PCR product of EGFP; 3: PCR of SV40polyA. Figure 2 shows overlapping PCR of IE1 and EGFP. Electropherogram; M: lOkb DNA marker; 1 : PCR product of IE 1 -EGFP;

Figure 3 is an electropherogram of the triplet gene IEl-EGFP-SV40polyA; M: 10 kb DNA marker; 1 : PCR product of IEl-EGFP-SV40 polyA;

Figure 4 is a PCR and double restriction analysis of the transfer vector pUC19-IEl-EGFP-SV40polyA; M: lOkb DNA marker; 1 : PCR product of pUC19-IEl-EGFP-SV40polyA; 2: pUC 19-IE 1 -EGFP- Double digestion product of SV40polyA;

Figure 5 is a PCR and double restriction map of the transfer vector pUC19-IEl-EGFP-SV40polyA-gp64; M: lOkb DNA marker; 1: PCR product of pUC19-IEl-EGFP-SV40polyA-gp64; 2: pUC19-IEl- Double digestion product of EGFP-SV40 polyA-gp64; Figure 6 is a PCR and double restriction analysis of the recombinant transfer vector UC 19-lef7-IE 1 -EGFP-S V40polyA-gp64; M: lOkb DNA marker; 1 : pUC 19-lef7-IE 1 -EGFP-S V40- a PCR product of gp64; 2: a double-cut product of pUC19-lef7-IEl-EGFP-SV40-gp64;

Figure 7 is a micrograph of normal cells of silkworm;

Figure 8 is a microscopic fluorescence diagram of silkworm cells transfected only with the recombinant transfer vector UC 19-lef7-IE 1 -EGFP-S V40 polyA-gp64;

Figure 9 is a microscopic fluorescence diagram of the co-transfection of the recombinant virus RBmNPV-EGFP genome with the recombinant transfer vector pUC 19-lef7-IE 1 -EGFP-S V40 polyA-gp64;

Figure 10 is a PCR and double restriction analysis of the recombinant plasmid pUC19-LEF7; M: lOkb DNA marker; 1: PCR product of pUC19-LEF7; 2: double digestion product of pUC19-LEF7;

Figure 11 is a PCR and double restriction analysis of the transfer vector pUC19-LEF7-GP64 without a marker gene; M: 10 kb DNA marker; 1 : PCR product of pUC19-LEF7-GP64; 2: Double enzyme of PUC19-LEF7-GP64 Cut the product. detailed description

It should be noted that the following detailed description is illustrative and further illustrative of the invention.

In the present specification, unless otherwise specified, the technical terms used are common terms used by those of ordinary skill in the art; the experimental methods in which no specific conditions are specified in the present specification are conventional experimental methods; the test materials used in the present specification are as follows. In particular, the products are purchased for sale. The composition and preparation method of various reagents and media can be found in the operation of the general experimental manual.

By analyzing and studying the BmNPV genome, it is speculated that about 50% of the genes of BmNPV are not necessary for the proliferation of the virus in cultured cells. The baculovirus genome can be deleted in larger regions, thereby increasing the effective pathway for expression of foreign genes. In addition, the stability of recombinant proteins can be enhanced by removing certain genes, such as cysteine, cathepsin, and chitinase genes. The present invention simultaneously knocks out cathepsin (cathepsin) The invention is illustrated by taking chitinase as an example.

Cathepsin and chitinase are late expression genes of BmNPV, which are adjacent to each other in the genome and have opposite transcriptional directions. They are non-essential genes for the replication of Bombyx mori nuclear polyhedrosis baculovirus. Related literature reports that there is no effect on the replication of viral genes after knockout.

The invention will be further described below in conjunction with the drawings and embodiments.

Example 1: Construction of transfer vector pUC 19-IE 1 -EGFP-SV40 polyA:

1. Recombinant plasmid PSK-IE1-EGFP constructed by Gao Zhen et al. (Gao Zhen, Hong Yeting, Zhou Fang et al. Cloning, expression analysis and isolation and identification of BmAGO1 gene from Bombyx mori [OL].

[2012-01-09]. The Chinese Science and Technology Thesis is on the line, htt: 〃 www.paper.edu.cn/index.php/default/releasepaper/content/201201 -252) for the template design:

(IE-l)F: 5-TTTGrCG, 4CAGTCGTTTGGTTGTTCACGATCG-3 (the bolded slanted portion is the Sail cleavage site)

(EGFP)F: 5-TTATGTCCCGAACTGCAAATCATGGTGAGCAAGGGCGAG-3

(EGFP) : 5-AATGTGGTATGGCTGATTATGATCTTACTTGTACAGCTCGTC-3 (SV40polyA) F: 5-

CATGGACGAGCTGTACAAGTAAGATCATAATCAGCCATACC-3

(SV40 polyA): 5-CGGGG 4CCATCCAGACATGATAAGATACATTG-3 (the cleavage site of Kpnl is shown in the bolded slope) (the PCR results of the three genes are shown in Figure 1), which is consistent with the expected size.

2. The triplet gene is spliced by overlapping PCR: IE1-EGFP-SV40 (as shown in Figure 2 and Figure 3), wherein the base sequence of IE1 is shown as SEQ ID NO: 1, and the base sequence of EGFP is SEQ. As shown by ID NO: 2, the base sequence of SV40polyA is shown in SEQ ID NO: 3:

(1) The PCR reaction system for IE1 and EGFP is as follows:

ddH ₂ 0 \2.5μΙ,

5 X Phusion HF Buffer 10 μ∑

lOXdNTP 7μ∑ PCR purification product of IE1 5μ1

PCR purification of EGFP 5μ1

Phusion hot start 0.5 μL

Total 40 μΐ

The PCR reaction procedure is as follows:

98 ° C pre-denaturation 3 min

98 °C denaturation 30 s - 58 °C annealing 30 s b 8 cycles

72°C extension 1 min30s―

72°C extension lOmin

(2) After the above reaction, a system of ΙΟμΙ^ is added to carry out the reaction:

Dd3⁄40 2 )

IE1 upstream primer 2 L

EGFP downstream primer 2 L

5 X phusion HF Buffer 4μ∑

The PCR reaction procedure is as follows:

98 ° C pre-denaturation 3 min

98 °C denaturation 30 s

Annealing at 58 °C 30 s >- 22 cycles

72 ° C extension 1 min30s

72°C extension lOmin

The IE1-EGFP ligation product was recovered by tapping and stored at -20 °C for use.

The method of joining the sub-gene of IE 1 -EGFP to SV40polyA is the same as the method described in the above (1) and (2).

(3) The double digestion system of the PCR product of the vector pUC19 and the triplet gene IE-EGFP-SV40polyA is as follows: In a sterile Eppendorf tube, add in order:

lOxFD Buffer 5 μL

IE-EGFP-S V40polyA/pUC 19 42^

Sal I 1.5

Kpnl 1 μL·

Total 50

The tea was digested at 37 ° C for 30 min, and the tapping was recovered for use.

(4) Connection reaction:

Ligation high 6 μL

IEl-EGFP-SV40polyA 4 μL

pUC19 2 μ

Total 12

After ligation at room temperature for 30 min, the ligation product of the transfer vector pUC19-IEl-EGFP-SV40polyA was obtained, and the subsequent experiments were continued after sequencing.

1) Take the 5 μ transfer vector pUC19-IEl-EGFP-SV40polyA ligation product, add 200 L DH5a competent cells (purchased from Invitrogen) suspension, mix gently, and place on ice for 30 min;

2) 42 bath heat shock for 90 seconds, quickly remove and cool on ice for 3-5 min;

3) Add 1 mL of pre-warmed LB liquid medium (peptone and yeast extract purchased from Oxoid) at 37 °C (without antibiotics), mix and shake at 37 °C for 1 h, and bring the bacteria back to normal. Growth state

4) Centrifuge the transformed bacterial solution at 4000 rpm for 10 min, discard the supernatant of l mL, and resuspend the pellet to take about 200 μΐ of the culture solution;

5) After standing for 30 min, the bacterial liquid is completely absorbed by the medium, and then inverted and protected from light for 16-24 hours;

6) Pick up the white colonies containing the recombinant plasmid, and carry out PCR and restriction enzyme digestion. The electrophoresis results are shown in Figure 4 (and sent to the Huada gene for subsequent experiments), indicating the transfer of the marker gene. Upon successful construction, the homologous sequences at both ends of any BmNPV replication non-essential gene can be cloned based on this vector.

Example 2: Construction of recombinant transfer vector UC 19-lef7-IE 1 -EGFP-S V40polyA-gp64

The BmNPV viral genome (in which the viral DNA extraction kit was purchased from Biotech Bioengineering Co., Ltd.) was used as a template to amplify the homologous sequences lef7 and gp64, and the genes lef7 and gp64 were cloned according to the procedures described in Example 1-2. In the transfer vector pUC19-IEl-EGFP-SV40polyA, a recombinant transfer vector UC 19-lef7-IE 1 -EGFP-SV40 polyA-gp64 was obtained.

Then, the recombinant transfer vector UC 19-lef7-IE 1 -EGFP-S V40polyA-gp64 was identified by PCR and double digestion. The electrophoresis results are shown in Figure 5 and Figure 6. The identification results in Figure 5 indicate the homology of one end of the target gene. The sequence was cloned into the transfer vector pUC19-IEl-EGFP-SV40polyA. The results of Fig. 6 showed that the homologous sequences at both ends of the target gene were successfully cloned into the transfer vector pUC19-IEl-EGFP-SV40polyA, and the sequence was correctly used for subsequent transformation. Dyeing experiment.

Example 3: Extraction of the BmNPV viral genome:

1) Collect about 100 ml of cell liquid infected by Bombyx mori nuclear polyhedrosis baculovirus, freeze-thaw three times at -80 °C, 37 °C, centrifuge at 1 °C for 1 h, and take the supernatant;

2) 4 °C, 30000 rpm over 1 h, discard the supernatant, collect virions, dissolve in 1 x TE, add 2 (^L, 20 mg/ml proteinase K (purchased from Invitrogen), 20μ, 10% SDS, 50 °C water bath overnight (at least 12h);

3) Add ΙΟμΙ^, lOmg/ml of RNaseA (purchased from Beijing Dingguo Biotechnology Co., Ltd.), 37 °C water bath lh;

4) Extract the viral genome with phenol and chloroform:

a, first gently mix with (equal volume) Tris-balanced phenol, continue for 5min, centrifuge at 12000rpm lOmin;

b. Reuse phenol, chloroform and isoamyl alcohol in a volume ratio of 25:24:1 (the sum of the three volumes) After the completion of the supernatant, the volume is the same) Extract once, gently mix, for 5min, 12000rpm, centrifugation lOmin;

c, using a volume ratio of 24: 1 chloroform and isoamyl alcohol (the sum of the volume is the same as the supernatant volume after the completion of the core), once centrifuged at 12000 rpm lOmin;

5) Take the supernatant, gently mix with 2 times the volume of the pre-cooled absolute ethanol in the supernatant after the heart is finished, add 1/10 volume to the supernatant after the heart is finished. NaAc, which does not include the volume of absolute ethanol, was precipitated at -80 ° C for 2 h, centrifuged at 12000 rpm for 30 min, and the precipitate was dissolved in ΙΟΟμΙ lx TE at 4 ° C for use.

Example 4: Bombyx mori nuclear polyhedrosis virus genome and recombinant transfer vector pUC19-lef7-IEl-EGFP-SV40 polyA-gp64 were co-transfected into BmN cells (purchased from Invitrogen) by liposome (purchased from Roche). :

1) Inoculate 1×10 ⁴ BmN cells in a 35 mm culture dish and incubate for 12 hours to allow the cells to grow as close as possible. When the cells grow to 60%-70% abundance, wash the cells with serum-free medium. 2-3 times in standby, prepare recombinant transfer vector pUC 19-lef7-IE 1 -EGFP-S V40polyA-gp64 (2-3 g) about 30μ1, wild Bombyx mori nuclear polyhedrosis virus genomic DNA (3-4 g) 30μ1, supplemented with 无μΙ with serum-free medium, which is liquid A;

2) Take ΙΟμΙ liposome and add it to 100 μΐ with serum-free medium, which is sputum;

3) Mix the sputum and sputum liquids evenly, let stand for 15-20 minutes at room temperature, add 800 μΐ of serum-free medium and mix well. Aspirate the medium in the plate with a pipette tip and add dropwise to the plate, 27 ° The incubator was cultured for 12 hours under C, and 20 ml of fetal bovine serum FBS medium (purchased from Invitrogen), 1 ml, was added for further 48 hours, and observed by an inverted fluorescence microscope, and the results showed that a few cells showed weak fluorescence ( See Figure 9); The silkworm cells of the silkworm normal cells and the recombinant transfer vector UC 19-lef7-IE 1 -EGFP-S V40polyA-gp64 were separately used as controls. Figure 7 shows a micrograph of normal silkworm cells, and Figure 8 shows a single transfection. Microscopic fluorescence of the silkworm cells of the recombinant transfer vector UC 19-lef7-IE 1 -EGFP-S V40 polyA-gp64. As time goes on, the fluorescence will gradually increase, indicating that the recombinant transfer vector and the viral genome are co-transfected successfully, and the marker gene replaces the target gene on the viral genome, thereby achieving the purpose of deleting the target gene.

Example 5: Screening of recombinant virus RBmNPV-EGFP with fluorescently labeled gene

1) When the fluorescence is gradually enhanced, the cells are fixed with 1% low melting point agarose, and the fluorescent cells are picked up by the sterilized gun head, and placed in a 96-well plate to expand the culture;

2) The viral genome was extracted from the cells obtained in the step 1) and identified.

Example 6: Recombinant virus RBmNPV-EGFP genome extraction

The recombinant virus RBmNPV-EGFP genome was extracted according to the method and procedure described in Example 3. Example 7: Construction of a marker-free gene transfer vector

The label-free gene transfer vector was constructed according to the method and procedure described in Example 2, and PCR and double enzyme digestion were performed. The electrophoresis results are shown in Figure 10, Figure 11, Figure 10, and Figure 11. The transfer vector of the marker-free gene was successfully constructed. The purpose of constructing this vector is to replace the marker gene on the viral genome so that the marker gene can be reused to continue knocking out other targeted genes.

Example 8: Recombinant virus RBmNPV-EGFP genome co-transfection with marker-free gene transfer vector and screening of recombinant virus RBmNPV

The genomic group of the recombinant virus RBmNPV-EGFP was co-transfected with the marker-free gene transfer vector and its recombinant virus RBmNPV according to the methods and procedures described in Examples 4-5.

The above description is only an embodiment of the present invention, and it should be noted that those skilled in the art can also make some improvements and retouching without departing from the core technical features of the present invention. And improvements are also within the scope of the patent protection of the present invention.

Claims

Claim

A method of producing a transfer vector with a marker gene, the method comprising the steps of:

(a) Preparation of a triplet gene by overlapping PCR: IEl-EGFP-SV40 polyA;

(b) The IEl-EGFP-SV40 polyA obtained in the step (a) was cloned into the vector pUC19 to obtain the transfer vector UC 19-IE 1 -EGFP-SV40 polyA.

The method according to claim 1, wherein the method further comprises:

(c) Transfer vector UC 19-IE 1 -EGFP-S V40 polyA transforms DH5a E. coli competent cells;

(d) Screening for blue and white spots on a solid medium containing ampicillin, and picking up white colonies containing the transfer vector pUC19-IEl-EGFP-SV40polyA;

(e) Transfer vector UC 19-IE 1 -EGFP-S V40 polyA for PCR and double enzyme digestion, and

The method according to claim 1, wherein the step (a) specifically comprises:

1) The genes are sequentially obtained by PCR: IE, EGFP, SV40polyA;

2) IE1 and EGFP were spliced by overlapping PCR method, and the ligated product of IE1-EGFP was used as a subunit to splice with the gene SV40polyA to obtain the triplet gene IEl-EGFP-SV40polyA.

A transfer vector carrying a marker gene, which is produced by the method according to any one of claims 1-3.

A method for knocking out a non-essential gene for knocking out a Bombyx mori nuclear polyhedrosis virus, the method comprising the steps of:

(1) Preparation of the homologous sequence of the silkworm nuclear polyhedrosis virus BmNPV replication non-essential fragment: lef7, gp64;

(2) The homologous sequences lef7 and gp64 obtained in the step (1) are sequentially cloned into the method of claim 2. In the transfer vector pUC19-IEl-EGFP-S V40polyA, recombinant transfer pUC 19-lef7-IE 1 -EGFP-S V40polyA-gp64 was obtained, and BmN cells were transfected with the silkworm nuclear polyhedrosis virus genome, and homologous recombination was obtained. Recombinant disease RBmNPV-EGFP with EGFP tag;

(3) Extraction of recombinant virus The genome of RBmNPV-EGFP was transfected with pUC19-lef7-gp64 into BmN cells;

(4) Screening for the recombinant virus RBmNPV without a marker gene.

The method according to claim 5, wherein the step (1) specifically comprises:

1) Extracting the BmNPV genome of the Bombyx mori nuclear polyhedrosis virus;

2) PCR method to obtain homologous sequences LEF7, gp64 at both ends of the replication non-essential fragment.

The method according to claim 5, wherein the recombinant transferor pUC19-lef7-IEl-EGFP-SV40polyA-gp64 and the Bombyx mori nuclear polyhedrosis virus gene are mediated by liposome in the step (2) BmN cells were co-transfected by transfection.