WO2022236720A1 - Method for knocking out essential gene of baculovirus/bacmid virus - Google Patents

Abstract

Provided is a method for knocking out an essential gene of a baculovirus, comprising the following steps: performing homologous recombination and purification on a transfer vector and a modified baculovirus/bacmid in silkworm cells, and extracting DNA; transforming DNA into the escherichia coli competent state, and selecting colonies for bacterial liquid PCR to obtain a strain containing an intermediate knockout strain; and then co-infecting the intermediate knockout strain and an unmodified bacmid virus with the silkworm cells, performing homologous recombination, and extracting DNA; and transforming DNA into the escherichia coli competent state, and selecting blue colonies for bacterial liquid PCR to obtain a strain containing the bacmid having the essential gene knocked out, so as to knock out the essential gene of the baculovirus.

Description

A method for knocking out essential genes of baculovirus technical field

The invention belongs to the field of insect baculovirus gene function research, and in particular relates to a method for knocking out an essential gene of the baculovirus, which can save labor and improve work efficiency.

Background technique

Baculoviruses are enveloped double-stranded circular DNA viruses with a genome size of 80-180 kb and 100-200 coding genes. According to the importance of function, genes can be divided into essential genes and non-essential genes. Baculoviruses are versatile and can be used for overexpression of exogenous genes, biopesticides, baculovirus surface display and gene therapy vectors. Bombyx mori Nuclear Polyhedrosis Virus (BmNPV) is a natural pathogenic microorganism of silkworm and a representative of baculovirus. The blood-type pustular disease caused by the virus has the characteristics of large outbreak scale and long-term impact. The annual loss of silkworm cocoons due to the disease accounts for more than 60% of the total silkworm disease loss. However, there is no specific medicine, and the main focus is on prevention. Studying the essential genes of baculovirus can analyze the replication and assembly mechanism of the virus, and provide a theoretical reference for anti-baculovirus.

To study the functions of essential genes, the knockout strains after knockout of essential genes must be obtained first. Since the baculovirus cannot replicate, assemble or transport after knocking out the essential genes, it cannot spread and spread among cells cultured in vitro. Therefore, homologous recombination in unmodified BmN cells cannot obtain purified knockout strains. The traditional method is to integrate the essential gene to be knocked out into the genome of BmN cells by transposition, then use the transgenic cell line to perform homologous recombination to knock out the essential gene, and use low melting point agarose for multiple rounds of purification. The disadvantage of this method is that it is time-consuming and labor-intensive to obtain transgenic cell lines, and the cells are prone to contamination. In addition, the purification process requires high technical requirements and a long cycle, and requires the use of expensive low-melting point agarose. There is also a method for knocking out essential genes in E. coli based on RED recombination technology. This method does not require the construction of transfer vectors. The recombined homology arms are directly designed on the primers, and then the linear PCR products are used to directly transform BW25113 E. coli. However, Due to the low efficiency of linear DNA transformation and the low recombination rate of RED technology, electroporation and antibiotic selection are necessary. In addition, because the PCR primers are very long, the cost is high and the amplification conditions need to be explored, which is time-consuming and labor-intensive. The PKD46 plasmid encoding RED recombinase uses a temperature-sensitive replicon, which needs to be cultured at 30°C, and high temperature treatment at 42°C is required to lose the PKD46 plasmid, while the expression of RED recombinase needs to be induced with L-arabinose. Therefore, although the knockout method based on RED technology does not require the construction of transfer vectors, it requires high technology and equipment and is not easy to implement.

Based on the above actual situation, there is an urgent need to develop a new method that can efficiently knock out the essential genes of baculovirus, does not require special equipment, and is easy to operate.

technical problem

The purpose of the present invention is to provide a method for knocking out essential genes of baculovirus, so as to obtain efficiently purified viruses knocked out of essential genes and improve work efficiency.

technical solution

In order to achieve the above-mentioned purpose of the invention, the technical scheme adopted in the present invention is.

A method for knocking out essential genes of baculovirus, comprising the following steps.

(1) Construction of transfer vector and transformation of baculovirus bacmid.

(2) Carry out homologous recombination and purification of the transfer vector and the transformed bacmid in silkworm cells, and extract the DNA.

(3) Transform the extracted DNA into competent Escherichia coli, pick colonies for bacterial liquid PCR, and obtain strains containing intermediate knockout strains.

(4) Co-infect silkworm cells with intermediate knockout virus and unmodified bacmid virus for homologous recombination and extract DNA.

(5) Transform the extracted DNA into competent Escherichia coli, pick blue colonies for bacterial liquid PCR, and obtain a strain containing the bacmid with the knockout of the essential gene, and realize the knockout of the essential gene of the baculovirus.

In the present invention, the essential gene is the orf92 gene; the silkworm cell is the silkworm ovary cell line (BmN); and the baculovirus is the silkworm nuclear polyhedrosis baculovirus (BmNPV).

In the present invention, in step (1), the amplified product of the essential gene sequence to be knocked out and its upstream and downstream sequences is connected to the linear carrier by enzyme cleavage to construct an intermediate transfer vector, and then the amplified product of the reporter gene is passed through the enzyme The transfer vector is constructed by cutting and connecting to the linear amplification product of the intermediate transfer vector; using baculovirus bacmid DNA as a template, PCR amplifies the necessary gene to obtain the necessary gene amplification product; then the necessary gene amplification product is connected to On the vector, the essential gene plasmid is constructed; then the essential gene is transposed to the polyhedron locus of the bacmid through the Bac-to-Bac transposition system to obtain the transformed bacmid. Step (1) specifically comprises the following steps: amplify the sequence of the essential gene to be knocked out and its upstream and downstream sequences by PCR, and simultaneously use reverse PCR to linearize the carrier, and construct an intermediate transfer vector by enzyme digestion ligation reaction; To PCR, the reporter gene partly replaces the gene to be knocked out; PCR amplifies the essential gene to be knocked out, and transposes the essential gene to the polyhedron locus through the Bac-to-Bac transposition system to obtain the gene to be knocked out The bacmid of the essential gene driven by the polyhedron promoter, that is, the transformed bacmid of the bacmid. According to the expression phase of the essential gene, the polyhedron promoter can be replaced by the IE1 immediate early promoter or the self-promoter of the essential gene to be knocked out by SnaBI and BamHI double digestion of the pFastbac1 plasmid.

In the present invention, the carrier is used as a template to carry out inverse PCR to linearize the carrier to obtain a linear carrier; the bacmid virus DNA is used as a template to amplify the essential gene and its upstream and downstream sequences by PCR to obtain the necessary gene sequence to be knocked out and its sequence. Amplified products of upstream and downstream sequences; using the intermediate transfer vector as a template, perform reverse PCR to obtain the linear amplification product of the intermediate transfer vector; use the reporter gene plasmid as a template, PCR amplify the reporter gene and its polyhedron promoter, and obtain the report Gene amplification product. The vector plasmid can be puc19 or some smaller plasmids; inverse PCR uses high-fidelity enzymes; by using inverse PCR, only two rounds of enzyme digestion and ligation reactions can be used to construct the transfer vector, while the traditional method requires three rounds Enzyme ligation reaction.

In the present invention, in step (2), the baculovirus bacmid co-transfected with the transfer vector and the transformation of the silkworm cell is subjected to homologous recombination; then the supernatant is incubated with the silkworm cell in a 96-well plate; Take out the supernatant from the wells with the largest number of green fluorescent cells, especially those with clusters of fluorescent cells, and add them to another 96-well plate for incubation; then take the supernatant from the wells with the largest number of green fluorescent cells and add them to the plate. DNA was extracted after cultured in cell flasks full of silkworm cells for 3 days. Co-transfect silkworm cells with the constructed transfer vector and the transformed baculovirus bacmid DNA, and then purify without using low-melting point agarose, which is one of the technical means to distinguish the prior art. Co-transfection with the modified baculovirus bacmid and the transfer vector eliminates the need to construct a cell line stably expressing the gene to be knocked out; by using a 96-well plate, the virus is purified by a dilution method without using low-melting point agarose.

In the present invention, in step (3), the DNA extracted in step (2) is transformed into competent cells (Escherichia coli DHOB), and then spread on the agar culture plate. Strains containing intermediate knockouts. Use the kit to extract bacmid DNA from BmN cells without ultra-high-speed centrifugation to collect virus particles, and without protein K digestion and phenol-chloroform extraction; especially, DH10B competent ( Conventional competent method), without the use of electric shock apparatus.

In the present invention, in step (4), the bacmid DNA is extracted from the strain containing the intermediate knockout strain and transfected into silkworm cells, then the supernatant is taken to co-infect the silkworm cells with the unmodified bacmid virus, and cultured for 3 days , directly extract DNA without purification.

In the present invention, in step (5), the DNA extracted in step (4) is transformed into competent cells, and then spread on an agar culture plate. After culturing, blue colonies are picked for bacterial liquid PCR to obtain rod-shaped The bacterial strain of the bacmid with virus essential gene knockout realizes the purpose of the invention.

The invention discloses a bacterial strain containing a bacmid knocked out of the essential baculovirus gene obtained according to the method for knocking out the essential gene of the baculovirus.

The invention discloses the application of the transformed baculovirus bacmid in knocking out the essential gene of the baculovirus; using the bacmid DNA of the baculovirus as a template, the essential gene is amplified by PCR to obtain an amplification product of the essential gene; The amplified product is connected to the carrier to construct the essential gene plasmid; then the essential gene is transposed to the polyhedron locus of the bacmid through the Bac-to-Bac transposition system to obtain the transformed bacmid.

In the present invention, the transformation, transfection, and infection conditions involved in step (1), step (2), step (3), step (4) and step (5) are consistent with conventional requirements, and the specific methods do not affect the technical effect of the present invention realization.

Step (1) of the present invention carries out two rounds of enzyme-cut ligation by two rounds of inverse PCR to construct the transfer vector, while the existing method needs to carry out successively the left arm enzyme-cut connection, the right arm enzyme-cut connection and the reporter gene enzyme-cut connection, a total of three steps: The transfer vector can be constructed by rounds of enzyme digestion and ligation reactions. Step (2) Co-transfect BmN cells with the transformed bacmid DNA and the transfer vector; use a 96-well plate for purification, which solves the problem of using low-melting point agarose in the prior art. In steps (3) and (5) of the present invention, there is no need for ultra-high-speed centrifugation (>35000rpm) to collect virus particles, and the virus DNA is extracted from BmN cells through a kit; the calcium chloride method is used for competence, and no electric shock apparatus is needed.

Beneficial effect

Due to the application of the above technical solutions, the present invention has the following advantages compared with the prior art.

1. The invention provides a method for efficiently constructing a transfer vector, which only needs two rounds of enzyme cleavage and ligation reactions, while the traditional method requires three rounds of enzyme cleavage and ligation reactions.

2. The method for purifying baculovirus in cultured cells disclosed by the invention uses a 96-well plate to purify the virus by diluting the virus, does not need to use low-melting point agarose, and is simple in operation and economical and efficient.

3. The method for knocking out the essential gene of baculovirus disclosed in the present invention is simple and efficient because it does not need to construct a cell line stably expressing the gene to be knocked out, and it does not need to use equipment such as electric shock apparatus and ultra-high-speed centrifugation. Efficient technology that is easy to use.

Description of drawings

Figure 1 is a diagram of the upstream and downstream genome structures of recombinant and non-recombined bacmid orf92.

Figure 2 is the electrophoresis diagram of the restriction enzyme digestion verification of the transfer vector.

Fig. 3 is the electrophoresis diagram of PCR verification products of various strains.

Fig. 4 is the culture result of transformed DH10B competent cells.

Fig. 5 is a transfection-infection diagram of the orf92 knockout strain and its rescue strain.

Embodiments of the present invention

The plasmids, low-melting point agarose, PCR reagents, and enzyme cutting reagents involved in the present invention are all conventional reagents in the art; there are other methods in the prior art that can obtain knockout strains of essential gene knockouts, and the inventiveness of the present invention lies in providing new The technical idea is to obtain knockout strains for knockout of essential genes simply and efficiently, which solves the problem that the existing technology requires cell lines stably expressing the essential genes to be knocked out, low melting point agarose, electroshock apparatus, and antibiotics as necessary technical means. Typical materials and equipment: Bombyx mori ovary cell lines BmN, DH5a and DH10B were used in the experiment. The calcium chloride used to prepare competent cells was purchased from Sigma; the transfection reagent was Invitrogen lipo 2000. The equipment used (such as centrifuges and ultra-clean benches) are routine laboratory equipment. The specific operation method/test method involved in the present invention is a conventional method in the field, such as the conditions involved in transformation, transfection, and extraction are consistent with the conventional requirements in the field, and the specific method does not affect the realization of the technical effect of the present invention.

The method for knocking out essential genes of baculovirus disclosed in the present invention is as follows.

(1) Construction of transfer vector and transformation of baculovirus bacmid.

(2) Carry out homologous recombination and purification of the transfer vector and the transformed bacmid in silkworm cells, and extract the DNA.

(3) Transform the extracted DNA into competent Escherichia coli, pick colonies for bacterial liquid PCR, and obtain strains containing intermediate knockout strains.

(4) Co-infect silkworm cells with intermediate knockout virus and unmodified bacmid virus for homologous recombination and extract DNA.

(5) Transform the extracted DNA into competent Escherichia coli, pick blue colonies for bacterial liquid PCR, and obtain a strain containing the bacmid with the knockout of the essential gene, and realize the knockout of the essential gene of the baculovirus.

The present invention first introduces a copy of the essential gene to be knocked out at the polyhedron gene locus through the Bac-to-Bac transposition system, and co-transfects BmN cells with the transformed bacmid and the transfer vector, and undergoes the first homologous recombination After purification, the white spot knockout strain with the essential gene knockout but containing the necessary gene copy at the polyhedron gene locus is called an intermediate knockout strain, and then the unmodified bacmid virus (that is, the bacmid that can form blue spots, the merchant The original BmNPV bacmid bought there, present in DH10B competent, the polyhedron locus is not inserted into the foreign gene) and the intermediate knockout strain virus in BmN cells for the second homologous recombination to replace the polyhedron locus A copy of the essential gene, and finally obtain the coeruleus knockout strain with the essential gene knockout and the polyhedron gene locus translocation site intact (that is, the polyhedron locus does not have an exogenous gene inserted to form a locus coeruleus knockout strain).

In the embodiment of the present invention, the same system and procedure are used to amplify the sequence (reverse PCR, PCR) used to construct the transfer vector as follows.

Amplification system (50 μl).

ddH2O _: 32 μl.

5× PrimeSTAR Buffer (Mg2+ Plus): 10 μl.

1× dNTP Mixture (2.5 mM each): 4 μl.

Primer 1 (concentration 10 μmol): 1 μl.

Primer 2 (concentration 10 μmol): 1 μl.

Template (plasmid 100 ng, bacmid 400 ng): 1 μl.

DNA polymerase (Takara PrimeSTAR HS DNA Polymerase): 1 μl.

Amplification procedure.

94°C for 5 minutes.

30 cycles: 98°C 10 sec; 55°C 15 sec, 72°C 1 min/kb.

72°C for 10 minutes.

1 hour at 4°C.

Bacterial solution PCR verification system (15 μl).

ddH2O: ₁₁ μl.

10×PCR Buffer (Mg 2+ plus): 1.5 μl.

1× dNTP Mixture (2.5 mM each): 0.6 μl.

Primer 1 (concentration 10 μmol): 0.3 μl.

Primer 2 (concentration 10 μmol): 0.3 μl.

Template (bacteria solution): 1 μl.

DNA polymerase (Takara PrimeSTAR HS DNA Polymerase): 0.3 μl.

orf92 primer validation PCR program.

Forward primer: 5'- CTG GGATCC GCCACCATGGAATGCCCGTTTCAGATTCAAG -3'.

Reverse primer: 5'- GCCCT GAATTC CTACAAATAATAGTTGTACTTGATGG-3').

program.

94°C for 5 minutes.

30 cycles: 94°C 30 sec; 60°C 30 sec, 72°C 1 min/kb.

72°C for 10 minutes.

1 hour at 4°C.

M13 primer sequence.

Forward primer: 5'-CGCCAGGGTTTTCCCAGTCACGAC-3'.

Reverse primer: 5′-AGCGGATAACAATTTCACACGGGA -3'.

program.

94°C for 5 minutes.

30 cycles: 94°C 30 sec; 55°C 30 sec, 72°C 1 min/kb.

72°C for 10 minutes.

1 hour at 4°C.

The present invention will be further described below in conjunction with embodiment.

Embodiment 1: The method for knocking out essential genes of baculovirus according to the present invention.

(1) Construction of the transfer vector.

Using puc19 as a template, design primers (forward primer: 5'- TAT GAATTC ACAATCTGCTCTGATGCCGC-3'; reverse primer: 5'- ATA GGATCC AATCATGGTCATAGCTGTTTCCTG -3') to linearize puc19 by inverse PCR to obtain a linear puc19 vector.

Using the bacmid DNA of silkworm nuclear polyhedrosis virus as a template, PCR amplifies the orf92 gene (essential gene) and its upstream and downstream sequences, see Figure 1 (forward primer: 5'-ATA GGATCC GACAATCCAGTCACGGACGAACATC-3'; reverse primer: 5'- ACACT GAATTC AACGCTGCCTGACGACCAGTCTATG -3') to obtain PCR amplification products.

According to conventional methods, Takara BamHI+EcoRI restriction endonuclease was used for the first round of digestion, and Sangon’s T4 ligase was used for ligation according to the instructions, and the amplified product was connected to the linear puc19 vector to construct an intermediate transfer vector.

Using the intermediate transfer vector as a template, design primers (forward primer: 5'- TCA AAGCTT GTCGTCTGTGGGCACGTACACAATC-3'; reverse primer: 5'- CTC CTGCAG CGCTACAGTAAACCGCTCCAGTCTC-3') for reverse PCR to obtain linear amplification of the intermediate transfer vector product.

Primers were designed (forward primer: 5'- GTA GAATTC ATGGTGAGCAAGGGCGAGGA-3'; reverse primer: 5'- GCG AAGCTT TTACTTGTACAGCTCGTCCATGC -3'), and the green fluorescent gene egfp was amplified by PCR using the pEGFP-N1 plasmid as a template, and Takara EcoRI +HindIII Restriction endonuclease digestion, and a ligation reaction with a synthetic T4 ligase to connect egfp to the EcoRI-HindIII site of the pFastBac1 vector, thereby constructing pFastBac-pohp-egfp.

Primers were designed (forward primer: 5'- TAT AAGCTT CATCGTTTGTTCGCCCAGGACTC-3'; reverse primer: 5'-GGC CTGCAG TTACTTGTACAGCTCGTCCATGC-3'), and the pFastBac-pohp-egfp plasmid was used as a template to PCR amplify the reporter gene egfp and its polygon promoter, and the amplified product of the reporter gene was obtained.

According to the conventional method, Takara HindIII+PstI restriction endonuclease was used for the second round of digestion, and Sangon’s T4 ligase was used to perform the ligation reaction according to the instructions, and the reporter gene amplification product was connected to the linear amplification product of the intermediate transfer vector. , to construct the final transfer vector. The electropherogram of enzyme digestion verification is shown in Figure 2. Lane 1 is DL-5000; the intermediate transfer vector is digested with Takara BamHI+EcoRI restriction endonucleases to generate two bands (see lane 2), one is about 2700 bp puc19 vector fragment, the other is 3454 bp The insert fragment was as expected; the final transfer vector used Takara Digestion with BamHI+EcoRI restriction endonuclease produces two bands (see lane 3), which is due to a BamHI restriction site in the polyhedrin promoter-egfp expression cassette (see Figure 1), and the insert fragment produces A 1873bp band (containing the left arm and egfp) and a 1944bp band (containing the polyhedron promoter, 135bp at the 5' end of orf92 and the right arm) were found. Since the two bands are similar in size, they are shown in the electropherogram Overlapped; the final transfer vector was Takara Digest with BamHI+PstI restriction endonuclease to generate 1113bp left arm and 754bp egfp (cloned in the EcoRI-HindIII site of pfastbac1), see lane 4; the final transfer vector is restricted by Takara PstI+HindIII enzymatic cleavage, yielding 959 EGFP gene expression cassette of BP (see lane 5). The final transfer vector was digested with Takara HindIII+EcoRI restriction enzymes to generate 1745 Right arm of bp (see lane 6). The results of enzyme digestion confirmed the successful construction of the final transfer vector.

Transformation of baculoviruses.

Bombyx mori nuclear polyhedrosis virus (BmNPV) bacmid DNA was used as a template to amplify orf92 gene (essential gene) by PCR (forward primer: 5'- CTG GGATCC GCCACCATGGAATGCCCGTTTCAGATTCAAG -3'; reverse primer: 5'- GCCCT GAATTC CTACAAATAATAGTTGTACTTGATGG -3') to obtain the amplified product of orf92; according to the conventional method, use Takara BamHI+EcoRI restriction endonuclease to digest, and use Sangon's T4 ligase to perform the ligation reaction according to the instructions, and connect orf92 to the BamHI of the pFastBac1 vector -EcoRI site, thereby constructing pFastBac-pohp-orf92. See Figure 3, Lane 10 for the validation results of BamHI+EcoRI digestion of pFastBac-pohp-orf92.

According to the instructions of Invitrogen's insect cell-baculovirus expression system, orf92 was transposed to the polyhedron locus of the bacmid of baculovirus (BmNPV) through the Bac-to-Bac transposition system to obtain the essential gene to be knocked out by the polyhedron The bacmid of bacmid driven by somatic promoter, that is, the bacmid of engineered baculovirus (BmNPV). Orf92 and M13 primers were used for verification. According to the instructions, the bacmid with successful transposition was verified with M13 primer, and the band size was 2300 bp+inserted exogenous gene size, since orf92 size is 1176, so the band size is 2300+1176=3476 bp, verify the results, as shown in Figure 3, lanes 2 (orf92 primer) and 3 (M13 primer).

(2) Homologous recombination and purification of baculovirus in silkworm cells.

Co-transfect BmN cells with the final transfer vector constructed in step (1) and the transformed BmNPV bacmid with transfection reagents by the standard method of the manual.

4 days after transfection, take 1 μl of transfected cell supernatant and add it to 20 ml of cell culture medium containing 10% fetal calf serum (TC-100 medium, produced by Sigma Company, containing ⁵ ×107 BmN cells), and then averagely added to 96-well plate for routine incubation for 3 days (no need to use low-melting point agarose), and then select the well from the 96-well plate that has the largest number of green fluorescent cells (there are sheets of cells that fluoresce) from the 96-well plate , pipette 0.1 microliter of its supernatant and add it to 20 milliliters of cell culture medium containing 10% fetal bovine serum (TC-100 medium, produced by Sigma Company, containing ⁵ ×107 BmN cells), and then add it to 96 wells The plate was routinely incubated for 3 days (no need to use low-melting point agarose), and then from the 96-well plate, select the well with the largest number of green fluorescent cells observed by naked eyes (there are sheets of cells that fluoresce), and the cell supernatant was aspirated until it was confluent. In a 25 cm ² culture flask of BmN cells, the cell culture medium (TC-100 medium, produced by Sigma Company) containing 10% fetal bovine serum was routinely cultured for 3 days.

(3) The baculovirus was purified by transforming Escherichia coli DH10B.

Bacmid DNA was extracted from the virus-infected BmN cells cultured in step (2) for 3 days using the Bacmid mini-extraction kit of Biyuntian baculovirus shuttle vector, and transformed into DH10B competent cells (excluding bacmid), coated with tetracycline (10 µg/ml), kanamycin (50 µg/ml), gentamicin (7 µg/ml) on the LB agar culture plate, cultivated at 37°C for 16 hours, picked the colonies for bacterial liquid PCR, and the primers were orf92 amplification primers and M13 primers. If homologous recombination occurs, a reporter gene inserted in orf92 at its own locus will produce a band of size 1545 bp, and since the polyhedron locus has a complete copy of the orf92 gene, it will also produce a band of size 1188 bp bands. Therefore, a bacmid that has not undergone homologous recombination will produce a band with a size of 1188 bp, while the colony that produced two bands contained an intermediate knockout strain, see Figure 3, lanes 4 (orf92 primer) and 5 (M13 primer).

(4) Baculovirus undergoes homologous recombination in silkworm cells.

Extract bacmid virus DNA from the strain containing the intermediate knockout strain (white colony) obtained in step (3) and transfect BmN cells with the transfection reagent by the standard method of the instruction manual. After 4 days, collect the cell supernatant and untreated BmN cells were co-infected with modified bacmid virus (BmNPV) (which can form coeruleus), cultured for 3 days, and virus-infected BmN cells were obtained.

(5) The baculovirus was purified by transforming Escherichia coli DH10B.

Use Biyuntian baculovirus shuttle vector bacmid mini-extraction kit to extract bacmid DNA from the virus-infected BmN cells obtained in step (4), and transform DH10B competent cells with standard heat shock method, and apply to containing Tetracycline (10 µg/ml), kanamycin (50 µg/ml), IPTG (40 µg/ml), X-gal (100 µg/ml) on LB agar culture plates, cultured at 37°C for 48 hours (as shown in Figure 4 ), pick the blue colonies for bacterial liquid PCR, and the primers are orf92 amplification primers and M13 primers. The PCR results of the blue colonies containing the orf92 knockout strain, see Figure 3, lanes 6 (orf92 primer) and 7 (M13 primer), a strain containing the bacmid of the essential gene knockout (that is, containing the orf92 knockout strain) was obtained .

Time statistics.

Time spent on transfer vector construction: 6 days.

The time for homologous recombination and purification of baculovirus in silkworm cells: 13 days.

Purification time of baculovirus by transforming Escherichia coli DH10B: 1 day.

The time for baculovirus to perform homologous recombination in silkworm cells: 7 days.

Purification time of baculovirus by transforming E. coli DH10B: 2 days.

Total time spent: 29 days.

Feasibility: A knockout strain of the essential gene orf92 was obtained.

Experimental verification.

In order to confirm that the phenotype of the orf92 knockout strain was caused by the deletion of orf92 rather than mutations at other sites, a rescue strain was constructed through the Bac-to-Bac system, that is, the polygonal expression of the orf92 knockout strain obtained above A copy of orf92 was introduced into the somatic gene locus, and the construction verification results are shown in Figure 3, lanes 8 (orf92 primer) and 9 (M13 primer), and the transfection-reinfection experiments of the orf92 knockout strain and the orf92 knockout rescue strain were performed. Since orf92 is an essential gene, after deletion, the virus cannot spread between cells, so green fluorescence is only observed in successfully transfected cells, see Figure 5; and the rescue strain recovered due to the introduction of a copy of orf92 at the polyhedron gene locus Infection can spread between cells, so green fluorescence can be observed in the cell community, see Figure 5. This verification result confirmed that the phenotype of the orf92 knockout strain was indeed caused by orf92 knockout.

Comparative experiment one.

On the basis of Example 1, if step (2) is adjusted to: co-transfect BmN cells with the final transfer vector constructed in step (1) and the unmodified BmNPV bacmid with transfection reagents by the standard method of the manual. For the rest, according to Example 1, the virus cannot spread between cells, and the purification experiment cannot be performed, so the experiment is terminated.

Because orf92 is an essential gene, after the deletion, the competitiveness of the knockout strain is very weak, and it is difficult to form a green fluorescent cell community. Most importantly, after dilution, there is generally only one virus in a single cell, and the knockout strain cannot Diffusion, so it is impossible to purify by dilution method; in theory, knockout is easy but the ratio of homologous recombination is extremely low, so the technical difficulty is purification, because the virus cannot spread after knockout of essential genes, the key of the present invention is the knockout of essential genes The deleted virus can also be purified, so that a knockout strain of the essential gene orf92 can be obtained. Moreover, the present invention constructs a transfer vector by reverse PCR, which saves a round of enzyme digestion and ligation reaction compared with the traditional method; purifies the virus by dilution method without using low-melting point agarose, and in addition, by transforming the bacmid, it can Knockout of essential genes. The method of the present invention has complete reproducibility, and the knockout strain of the essential gene orf92 can be obtained through repeated experiments, and it is indeed the orf92 knockout strain through experimental verification.

The existing conventional methods are used for comparison, which are briefly described as follows. For more specific methods, please refer to any existing literature.

Comparative Example 1: A knockout method using a cell line stably expressing the gene to be knocked out.

(1) Establishment of cell lines stably expressing orf92 gene.

Use Piggybac to transpose the orf92 gene into the genome of silkworm cells, and then select by antibiotics to enrich the cells that have successfully transposed the orf92 gene.

(2) Construction of the transfer vector.

Design primers, PCR amplify the homologous recombination left arm of the orf92 gene, clone it to puc19, then PCR amplify the homologous recombination right arm of the orf92 gene, and clone it to the puc19 that has been connected to the left arm, and finally PCR amplification report The gene and its promoter are then cloned between the left and right arms to construct the final transfer vector.

(3) Homologous recombination and purification of baculovirus in silkworm cells.

Co-transfect BmN cells with the transfer vector constructed in (2) and BmNPV DNA using a transfection reagent. Six rounds of purification were performed using the method of low-melting point agarose; the existing purified viruses are covered with low-melting point agarose on the cells, so that the virus will not spread randomly with the liquid medium. This is because the low-melting point agarose is solid, and the virus can only slowly diffuse out from one point, so that the virus can be purified.

Time statistics.

Establishment of cell lines stably expressing orf92 gene: 35 days.

Time spent on transfer vector construction: 10 days.

The time for homologous recombination and purification of baculovirus in silkworm cells: 30 days.

Total time spent: 75 days.

Feasibility: A knockout strain of the essential gene orf92 was obtained.

Comparative Example 2: A knockout method using Red recombination technology in Escherichia coli.

(1) Amplification of linear PCR products.

Primers containing homology arms of the orf92 gene were designed, and PCR amplification was performed using chloramphenicol as a template, and gel recovery was performed.

(2) The linear PCR knockout fragment was transformed into BW25113.

Transform BW25113 competent cells with standard heat shock procedure; culture at 30°C, induce the expression of RED recombinase with L-arabinose, and heat shock at 42°C for half an hour to lose the pKD46 plasmid encoding RED recombinase.

Time statistics.

Amplification time of linear PCR product: 5 days.

The transformation time of linear PCR knockout fragment into BW25113: 2 days.

Total labor: 7 days.

Feasibility: No knockout strains were obtained.

Example Data Analysis.

The above three groups of data show that: compared with Comparative Example 1, the method of the present invention takes less time, which is 38.67% of the time spent in Comparative Example 1 (the knockout method using a cell line stably expressing the gene to be knocked out), and does not require expensive low-cost Melting point agarose; because the comparative example 2 (knockout method using Red recombination technology in E. coli) uses linear PCR fragments, the transformation efficiency is low, and the recombination rate of RED recombination technology is low. Knockout strains were obtained. In addition, the technology of the present invention is easy to operate without special equipment.

The present invention uses inverse PCR to construct an intermediate transfer carrier, and then uses inverse PCR to connect the reporter gene to the transfer carrier; then uses the transformed bacmid and transfer carrier to co-transfect BmN cells; dilutes the virus for purification without Use low-melting point agarose; then use the kit to extract bacmid DNA from transfected BmN cells, and transform Escherichia coli DH10B competent; then co-infect the obtained intermediate knockout virus with unmodified bacmid virus BmN cells do not need to be purified; finally, bacmid DNA was extracted from the transfected BmN cells using the kit, and transformed into competent E. coli DH10B to obtain a knockout strain of the essential gene orf92 knockout. The present invention does not need other steps, low-melting point agarose, electroshock apparatus, antibiotics and other technical means considered necessary in the prior art, and obtains the knockout strain of the essential gene orf92 knockout at low cost and high efficiency.

Claims (10)

A method for knocking out essential genes of baculovirus, comprising the following steps: (1) Construction of transfer vector and transformation of baculovirus bacmid; (2) Homologous recombination and purification of the transfer vector and the transformed baculovirus bacmid in silkworm cells, and DNA extraction; (3) Transform the extracted DNA into competent Escherichia coli, pick colonies for bacterial liquid PCR, and obtain strains containing intermediate knockout strains; (4) Co-infect silkworm cells with intermediate knockout virus and unmodified bacmid virus for homologous recombination and extract DNA; (5) Transform the extracted DNA into competent Escherichia coli, pick blue colonies for bacterial liquid PCR, and obtain a strain containing the bacmid with the knockout of the essential gene, and realize the knockout of the essential gene of the baculovirus. The method for knocking out the essential gene of baculovirus according to claim 1, characterized in that: the essential gene is the orf92 gene; the silkworm cell is the silkworm ovary cell line; and the baculovirus is the silkworm nuclear polyhedrosis baculovirus. The method for knocking out the essential gene of baculovirus according to claim 1, characterized in that: in step (1), the amplified product of the essential gene sequence to be knocked out and its upstream and downstream sequences to be knocked out is connected to the linear vector by restriction enzyme digestion above, construct an intermediate transfer vector, and then connect the reporter gene amplification product to the linear amplification product of the intermediate transfer vector by restriction enzyme digestion to construct the transfer vector; using the bacmid of baculovirus as a template, PCR amplifies the necessary genes to obtain Essential gene amplification product; then the essential gene amplification product is connected to the carrier to construct the essential gene plasmid; then the essential gene is transposed to the polyhedron locus of the bacmid by the Bac-to-Bac transposition system , to obtain the transformed baculovirus bacmid. The method for knocking out the essential gene of baculovirus according to claim 3, characterized in that: using the carrier as a template, carry out reverse PCR to linearize the carrier to obtain a linear carrier; using the baculovirus bacmid DNA as a template, PCR amplification Increase the essential gene and its upstream and downstream sequences to obtain the amplification product of the essential gene sequence and its upstream and downstream sequences to be knocked out; use the intermediate transfer vector as a template to perform reverse PCR to obtain the linear amplification product of the intermediate transfer vector; use the reporter gene The plasmid is used as a template, and the reporter gene and its polyhedron promoter are amplified by PCR to obtain the reporter gene amplification product. The method for knocking out the baculovirus essential gene according to claim 1, is characterized in that: in step (2), carry out homologous recombination in the co-transfection silkworm cell of the baculovirus bacmid DNA of transfer vector and transformation; Then Take the supernatant and incubate with silkworm cells in a 96-well plate; then remove the supernatant from the well with the largest number of green fluorescent cells and add it to another 96-well plate for incubation; then remove the supernatant from the well with the largest number of green fluorescent cells The supernatant was added to a culture flask covered with silkworm cells, and DNA was extracted after cultivation. The method for knocking out baculovirus essential genes according to claim 1, characterized in that: in step (3), the extracted DNA is transformed into Escherichia coli competent, then spread on the agar culture plate, after culturing, pick The bacterial colonies were taken for bacterial liquid PCR to obtain a strain containing an intermediate knockout strain. The method for knocking out the essential gene of baculovirus according to claim 1, is characterized in that: in step (4), extract the bacmid DNA of baculovirus from the bacterial strain containing intermediate knockout strain and transfect the silkworm cell, and then The supernatant was taken to co-infect the silkworm cells with the unmodified bacmid virus, cultured for 3 days, and then the DNA was extracted. The method for knocking out baculovirus essential genes according to claim 1, characterized in that: in step (5), the extracted DNA is transformed into Escherichia coli competent, then spread on the agar culture plate, after culturing, pick Take the blue colonies for bacterial liquid PCR to obtain a bacterial strain containing the bacmid knockout of the essential gene of baculovirus. The bacmid strain containing the knockout of the essential baculovirus gene obtained according to the method for knocking out the essential gene of the baculovirus according to claim 1. The application of the modified bacmid in the knockout of the essential gene of the baculovirus; using the bacmid DNA of the baculovirus as a template, the essential gene is amplified by PCR to obtain the amplification product of the essential gene; then the amplification product of the essential gene is connected to the carrier to construct the essential gene plasmid; then the essential gene is transposed to the polyhedron locus of the bacmid through the Bac-to-Bac transposition system to obtain the transformed bacmid.