Bac-to-Bac baculovirus expression system, recombinant stem grain, recombinant baculovirus and application thereof

Abstract

The invention discloses a Bac-to-Bac baculovirus expression system, a recombinant baculosome, a recombinant baculovirus and an application of the recombinant baculovirus. The Bac-to-Bac baculovirus expression system comprises a donor plasmid and a DH10Bac competent bacterium, and is characterized in that the donor plasmid is a recombinant plasmid; the donor plasmid comprises a first temperature-sensitive replicon control element and a heterologous functional gene segment expression cassette; the DH10Bac competent bacteria comprise a baculovirus genome plasmid bacmid and an auxiliary plasmid, and the auxiliary plasmid comprises a second temperature-sensitive replicon control element and a Tn7 transposase expression cassette; the first temperature-sensitive replicon control element and the second temperature-sensitive replicon control element are different. The recombinant bacilli are prepared on the basis of the Bac-to-Bac baculovirus expression system. The recombinant baculovirus is obtained by transfecting the recombinant baculovirus into insect cells and culturing the recombinant baculovirus. The recombinant plasmid prepared by the invention is purer and is not polluted by helper plasmids and donor plasmids.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a Bac-to-Bac baculovirus expression system, a recombinant bacmid, a recombinant baculovirus and applications thereof. Background Art

[0002] Insect baculovirus expression systems such as Bac-to-Bac and MultBac are widely used in heterologous protein expression, vaccine and antibody production, and large-scale preparation of recombinant adeno-associated viruses. The above baculovirus expression systems all contain baculovirus shuttle plasmids (bacmid), helper plasmids (Helper) and donor plasmids (Donor). With the help of the helper plasmid, the donor plasmid and the shuttle plasmid are recombined to obtain a recombinant bacmid containing an exogenous gene expression frame. In this process, the helper plasmid and the donor plasmid play a role by providing transposase and donor DNA fragments respectively. After recombination, these two plasmids become invalid plasmids. However, the recombinant bacmid prepared by this method is a mixed bacmid containing an invalid plasmid, the transfection efficiency is not high, and it is easy to recombine in the cell. By combining the mutated RK2 plasmid replicon with the donor plasmid and helper plasmid of the Bac-to-Bac system, temperature-sensitive donor plasmids and helper plasmids are constructed, and pure recombinant bacmids without interfering plasmids can be prepared (A recombinant bacmid and its preparation method, application number: 202310643354.7).

[0003] However, although the above temperature-sensitive donor plasmid and helper plasmid can be used to prepare pure recombinant bacmid, this method still has certain defects. The replicons of the above temperature-sensitive donor plasmid and helper plasmid are from the same source, both of which are RK2 plasmids. Due to the principle of plasmid incompatibility, the two are prone to mutual exclusion when they coexist in bacteria, affecting the copy number of the plasmid and ultimately affecting the efficiency of bacmid recombination. Summary of the invention

[0004] In view of the above defects of the prior art, the object of the present invention is to provide a Bac-to-Bac baculovirus expression system, a recombinant bacmid, a recombinant baculovirus and applications thereof, so as to solve the problem of mutual influence between temperature-sensitive donor plasmids and helper plasmids, and to prepare pure recombinant bacmids.

[0005] The specific technical solutions of the present invention are as follows:

[0006] The invention provides a Bac-to-Bac baculovirus expression system, which comprises a donor plasmid and a DH10 Bac competent bacterium; the donor plasmid comprises a first temperature-sensitive replicon control element and a heterologous functional gene fragment expression frame, the first temperature-sensitive replicon control element comprises a first replicon and a regulatory element of the first replicon; the DH10 Bac competent bacterium comprises a baculovirus genome plasmid bacmid and an auxiliary plasmid, the auxiliary plasmid comprises a second temperature-sensitive replicon control element and a Tn7 transposase expression frame, the second temperature-sensitive replicon control element comprises a second replicon and a regulatory element of the second replicon; the first temperature-sensitive replicon control element and the second temperature-sensitive replicon control element are different.

[0007] Further, the first temperature-sensitive replicon control element is selected from a temperature-sensitive pSC101 plasmid, the temperature-sensitive pSC101 plasmid is obtained by mutation of the RepA protein sequence of the pSC101 plasmid, and the first temperature-sensitive replicon control element is composed of ori and RepA gene expression cassettes of the temperature-sensitive pSC101 plasmid;

[0008] Preferably, the sequence of the first temperature-sensitive replicon control element is shown as SEQ ID NO.1.

[0009] Further, the second temperature-sensitive replicon control element is selected from a temperature-sensitive RK2 plasmid, the temperature-sensitive RK2 plasmid is obtained by mutation of the trfA protein sequence of the RK2 plasmid, and the second temperature-sensitive replicon control element is composed of oriV and trfA gene expression cassettes of the temperature-sensitive RK2 plasmid;

[0010] Preferably, the trfA protein sequence mutation of the RK2 plasmid is 235G, 163C, 171W, 173K, 203L, 213S, 230V, 247C, 254D, 313L, 316V, 332F or 374 deletion mutation.

[0011] Preferably, the sequence of the second temperature-sensitive replicon control element is shown as SEQ ID NO.2.

[0012] Furthermore, the donor plasmid is obtained by replacing pFast.Bac.Dual, pFastBac TM 1 or pFastBac TM The pUC ori in the HTA plasmid was replaced with the first temperature-sensitive replicon control element to construct;

[0013] Preferably, the donor plasmid is constructed by replacing the pUC ori in the pFast.Bac.Dual plasmid with the first temperature-sensitive replicon control element.

[0014] Furthermore, the helper plasmid is constructed by replacing pUC ori in the pMON7124 plasmid with a second temperature-sensitive replicon control element.

[0015] Furthermore, the heterologous functional gene fragment expression frame in the donor plasmid is selected from a GFP expression frame.

[0016] The present invention also provides a recombinant bacmid, which is prepared based on the Bac-to-Bac baculovirus expression system, specifically: a donor plasmid is transferred into DH10 Bac competent bacteria, blue-white screening is performed on a culture plate containing kanamycin, gentamicin, tetracycline, X-gal and IPTG, white colonies are picked, positive recombinant bacteria containing the recombinant bacmid are obtained after identification, and the recombinant bacmid is extracted.

[0017] The present invention also provides a recombinant baculovirus, wherein the recombinant bacmid is transfected into insect cells for culture, and the recombinant baculovirus is harvested from the culture.

[0018] The present invention also provides the use of the Bac-to-Bac baculovirus expression system, the recombinant bacmid or the recombinant baculovirus in preparing heterologous proteins.

[0019] The present invention has the following advantages and beneficial effects:

[0020] The present invention provides a Bac-to-Bac baculovirus expression system in which a donor plasmid and a helper plasmid can be controlled by temperature and their replication does not affect each other. The recombinant bacmid prepared based on the Bac-to-Bac baculovirus expression system is purer, free of contamination by the helper plasmid and the donor plasmid, and has a higher transfection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is a flow chart of the method for preparing the recombinant bacmid of the present invention;

[0022] Figure 2A Schematic diagram of the donor plasmid strategy for preparing recombinant bacmid;

[0023] Figure 2B Schematic diagram of the helper plasmid strategy for preparing recombinant bacmid;

[0024] Figure 2C Schematic diagram of the strategy for preparing recombinant bacmids;

[0025] Figure 3 Example 2 contains pFD pSC101-ts -Graph showing the growth effect of DH5α bacteria carrying GFP plasmid at different temperatures.

[0026] Figure 4The pMON7124 in Example 2 RK2-ts Growth effect diagram of DH5α bacteria carrying plasmid at different temperatures.

[0027] Figure 5 This is a diagram of the recombinant bacmid DNA extracted by pulsed-field gel electrophoresis in Example 2.

[0028] Figure 6 This is a fluorescence imaging image of the recombinant bacmid transfected into insect cells 3 days after Example 2. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as the specifications do not conflict with each other.

[0030] Example 1: Bac-to-Bac baculovirus expression system

[0031] The invention provides a Bac-to-Bac baculovirus expression system, which comprises a donor plasmid and a DH10 Bac competent bacterium; wherein the donor plasmid comprises a first temperature-sensitive replicon control element and a heterologous functional gene fragment expression frame, and the first temperature-sensitive replicon control element comprises a first replicon and a regulatory element of the first replicon; the DH10 Bac competent bacterium comprises a baculovirus genome plasmid bacmid and an auxiliary plasmid, and the auxiliary plasmid comprises a second temperature-sensitive replicon control element and a Tn7 transposase expression frame, and the second temperature-sensitive replicon control element comprises a second replicon and a regulatory element of the second replicon; and the first temperature-sensitive replicon control element and the second temperature-sensitive replicon control element are different.

[0032] In a specific embodiment, the first temperature-sensitive replicon control element is selected from the temperature-sensitive pSC101 plasmid, which is a low-copy plasmid mainly comprising an origin of replication (ori) and a long coding sequence encoding 316 amino acids (RepA protein). The RepA protein controls the number of plasmids by acting in trans on the ori region. When a mutation occurs at a specific amino acid site of the RepA protein, the RepA protein is inactivated at high temperatures, so that the plasmid cannot replicate and is gradually lost as the bacteria are propagated. The mutation of the RepA protein sequence causes the pSC101 plasmid to become sensitive to temperature, i.e., a temperature-sensitive plasmid. In the embodiment, the nucleotide sequences of the ori and RepA genes in the temperature-sensitive pSC101 plasmid are selected as replicons, and their sequences are shown in SEQ ID NO.1.

[0033] In a specific embodiment, the heterologous functional gene fragment expression frame is a green fluorescent protein (GFP) expression frame.

[0034] In one embodiment, a donor plasmid containing a pSC101 temperature-sensitive replicon (pSC101-ts) and a GFP expression cassette is constructed, specifically comprising: using a pFast.Bac.Dual (pFD) donor plasmid in the baculovirus expression system Bac-to-Bac, connecting the GFP exogenous gene expression cassette GOI with the pFD backbone plasmid by homologous recombination to construct a pFD-GOI donor plasmid, amplifying ori and RepA sequences in the temperature-sensitive pSC101 plasmid by PCR, and constructing a pFD-GOI donor plasmid by homologous recombination. pSC101 -ts -GOI temperature-sensitive donor plasmid, see attached Figure 2A .

[0035] In one embodiment, the second temperature-sensitive replicon control element is selected from the temperature-sensitive RK2 plasmid, and the protein expressed by the trfA gene on the RK2 plasmid acts in trans on the oriV region to control the replication of the plasmid (Haugan et al., 1992). The mutated trfA protein (such as the 235G synonymous mutation) is sensitive to temperature and has the characteristic of not being expressed at high temperatures (Karunakaran et al., 1998). Therefore, when the ambient temperature is too high, the RK2 plasmid in the bacteria will be gradually lost. In order to achieve the temperature sensitivity of the auxiliary plasmid, the nucleotide sequences of the oriV and trfA genes in the temperature-sensitive RK2 plasmid are selected as replicons in the embodiment, and its sequence is shown in SEQ ID NO.2.

[0036] In one embodiment, constructing a helper plasmid containing the RK2 temperature-sensitive replicon and the Tn7 transposase gene expression cassette specifically comprises: using PCR to amplify the oriV and trfA sequences in the temperature-sensitive RK2 plasmid, using the pMON7124 helper plasmid in the baculovirus expression system Bac-to-Bac, and constructing pMON7124 by homologous recombination. RK2-ts Temperature-sensitive helper plasmid, see attached Figure 2B .

[0037] In one embodiment, the DH10 Bac competent bacteria contain the baculovirus genome plasmid bacmid and pMON7124 RK2-ts Temperature-sensitive helper plasmid (DH10 Bac RK2-ts competent bacteria).

[0038] In one embodiment, a recombinant bacmid containing an exogenous gene expression cassette GOI is constructed. The strategy is shown in the attached Figure 2C , specifically: pFD pSC101-ts -GOI temperature-sensitive donor plasmid was electroporated into DH10 Bac RK2-ts Competent bacteria (containing the baculovirus genome plasmid bacmid and the temperature-sensitive auxiliary plasmid pMON7124 RK2-ts ). SOC medium was added and cultured at 30°C for 6 hours, then diluted and spread on a plate containing Kan + / Gen + / Tet + / IPTG / X-Gal plates were used for blue-white screening (cultured at 30°C). After 48 hours, white colonies were picked for colony PCR. After identification, positive recombinant bacteria containing recombinant bacmid were obtained. See attached Figure 2C .

[0039] In one embodiment, extracting the recombinant bacmid: extracting the recombinant bacmid without the helper plasmid and the donor plasmid. + The cells were shaken in an antibiotic medium at 37°C for 14 hours, and the recombinant bacmid was extracted using a plasmid extraction kit. The extracted plasmid was identified by pulsed field gel electrophoresis, and the results showed that the extracted plasmid contained only the recombinant bacmid, and contained no or very little auxiliary plasmid and donor plasmid.

[0040] In one embodiment, the recombinant bacmid is transfected into insect cells to obtain baculovirus. The recombinant bacmid extracted above is transfected into insect cells cultured on a wall, and the supernatant is collected by centrifugation after 3 days, which is the recombinant baculovirus. Fluorescence imaging results show that green fluorescent protein can be efficiently expressed in insect cells.

[0041] Example 2: Preparation of pure recombinant bacmids without interfering plasmids

[0042] The flow chart of the method for preparing the recombinant bacmid of the present invention is as follows: Figure 1 shown.

[0043] (1) Preparation of temperature-sensitive Bac-to-Bac donor plasmid and helper plasmid

[0044] The pFast.Bac.Dual (pFD) donor plasmid in the baculovirus expression system Bac-to-Bac was used. In the example, a green fluorescent protein (GFP) expression cassette was selected, and transcription was initiated by a CMV promoter. The GFP exogenous gene expression cassette was connected to the pFD backbone plasmid by homologous recombination to construct the pFD-GFP donor plasmid. In the example, the nucleotide sequences of the ori and RepA genes in the temperature-sensitive pSC101 plasmid were selected as plasmid replicons, and the sequence thereof is shown in SEQ ID NO.1. The ori and RepA sequences were amplified by PCR, and the pFD was constructed by homologous recombination. pSC101-ts -GFP temperature-sensitive donor plasmid, see attached Figure 2A . pSC101-ts -GFP temperature-sensitive donor plasmid was cultured at 30°C and 37°C (medium without antibiotics) for 12 hours, and then diluted 10,000 times and spread on a plate containing ampicillin resistance (Amp + ) plates, and then cultured overnight in incubators at 30°C and 37°C. pSC101-ts -GFP temperature-sensitive donor plasmid bacteria cannot grow under high temperature conditions, see attached Figure 3 .

[0045] The pMON7124 auxiliary plasmid in the baculovirus expression system Bac-to-Bac was used. In the example, the nucleotide sequences of oriV and trfA genes in the temperature-sensitive RK2 plasmid were selected as replicons, and the sequence thereof is shown in SEQ ID NO.2. The oriV and trfA sequences were amplified by PCR, and pMON7124 was constructed by homologous recombination. RK2-ts Temperature-sensitive helper plasmid, see attached Figure 2B . RK2-ts The bacteria carrying the temperature-sensitive helper plasmid were cultured at 30°C and 37°C (in a medium without antibiotics) for 12 hours, and then diluted 10,000 times and spread on a plate containing tetracycline resistance (Tet + ) plates, and then cultured overnight in incubators at 30°C and 37°C. RK2-ts Bacteria with temperature-sensitive helper plasmids cannot grow under high temperature conditions, see attached Figure 4.

[0046] (2) Preparation of recombinant bacmid

[0047] Construct a recombinant bacmid containing a GFP exogenous gene expression cassette. The strategy is shown in the attached Figure 2C . pSC101-ts -GFP temperature-sensitive donor plasmid was electroporated into DH10 Bac RK2-ts Competent bacteria (containing the baculovirus genome plasmid bacmid and the temperature-sensitive auxiliary plasmid pMON7124 RK2-ts ). SOC medium was added and cultured at 30°C for 6 hours, then diluted and spread on a plate containing kanamycin (Kanamycin + ) / Gentamicin (Gen + ) / Tet + / IPTG / X-Gal plates were used for blue-white screening (cultured at 30°C), and white colonies were picked after 48 hours for colony PCR. After identification, positive recombinant bacteria containing recombinant bacmid were obtained.

[0048] (3) Extraction of recombinant bacmid

[0049] Extract the recombinant bacmid without the helper plasmid and the donor plasmid. Inoculate the above positive recombinant bacteria in a culture medium containing only Kan + The cells were shaken in an antibiotic medium at 37°C for 14 hours, and the recombinant bacmid was extracted using a plasmid extraction kit. The extracted bacmid was identified by pulsed field gel electrophoresis. The results showed that the extracted bacmid contained only the recombinant bacmid, and contained no or very little interfering plasmids such as auxiliary plasmids and donor plasmids. Figure 5 .

[0050] (4) The above recombinant bacmid was -Insect ReagentMirus transfection reagent (ratio of 1 μg plasmid / 5 μL transfection reagent) was added to the adherent cultured ExpiSf9 insect cells (cell number was 2*10 6 / well), and fresh culture medium was replaced after 24 hours, and fluorescence expression was observed every day. The supernatant was collected after 72 hours, which was the recombinant baculovirus. The fluorescence imaging results showed that a large amount of green fluorescent protein was expressed in the cells, see Appendix Figure 6 .

[0051] From the above data, the baculovirus production system based on the dual temperature-sensitive replicon can avoid the problem of incompatibility between the donor plasmid and the helper plasmid, and effectively obtain pure recombinant bacmids without interfering plasmids such as the donor plasmid and the helper plasmid.

[0052] SEQ ID NO.1:

[0053]

[0054] SEQ ID NO.2:

[0055]

[0056] The specific embodiments described above are only preferred implementations of the present invention. It should be pointed out that a person skilled in the art can make several improvements or substitutions without departing from the principles of the present invention. These improvements or substitutions should also be regarded as within the scope of protection of the present invention.

Claims

1. A Bac-to-Bac baculovirus expression system, characterized in that: It comprises a donor plasmid and a DH10 Bac competent bacterium; the donor plasmid comprises a first temperature-sensitive replicon control element and a heterologous functional gene fragment expression frame, the first temperature-sensitive replicon control element comprises a first replicon and a regulatory element of the first replicon; the DH10Bac competent bacterium comprises a baculovirus genome plasmid bacmid and an auxiliary plasmid, the auxiliary plasmid comprises a second temperature-sensitive replicon control element and a Tn7 transposase expression frame, the second temperature-sensitive replicon control element comprises a second replicon and a regulatory element of the second replicon; The first temperature-sensitive replicon control element and the second temperature-sensitive replicon control element are different.

2. The Bac-to-Bac baculovirus expression system according to claim 1, characterized in that The first temperature-sensitive replicon control element is selected from a temperature-sensitive pSC101 plasmid, the temperature-sensitive pSC101 plasmid is obtained by mutation of the RepA protein sequence of the pSC101 plasmid, and the first temperature-sensitive replicon control element is composed of the ori and RepA gene expression cassettes of the temperature-sensitive pSC101 plasmid; Preferably, the sequence of the first temperature-sensitive replicon control element is shown as SEQ ID NO.

1.

3. The Bac-to-Bac baculovirus expression system according to claim 1, characterized in that The second temperature-sensitive replicon control element is selected from a temperature-sensitive RK2 plasmid, the temperature-sensitive RK2 plasmid is obtained by mutation of the trfA protein sequence of the RK2 plasmid, and the second temperature-sensitive replicon control element is composed of oriV and trfA gene expression cassettes of the temperature-sensitive RK2 plasmid; Preferably, the trfA protein sequence mutation of the RK2 plasmid is 235G, 163C, 171W, 173K, 203L, 213S, 230V, 247C, 254D, 313L, 316V, 332F or 374 deletion mutation.

4. The Bac-to-Bac baculovirus expression system according to claim 1, characterized in that The sequence of the second temperature-sensitive replicon control element is shown in SEQ ID NO.

2.

5. The Bac-to-Bac baculovirus expression system according to claim 1, characterized in that The donor plasmid was prepared by combining pFast.Bac.Dual, pFastBac TM 1 or pFastBac TM The pUC ori in the HTA plasmid was replaced with the first temperature-sensitive replicon control element to construct; Preferably, the donor plasmid is constructed by replacing the pUC ori in the pFast.Bac.Dual plasmid with the first temperature-sensitive replicon control element.

6. The Bac-to-Bac baculovirus expression system according to claim 1, wherein The helper plasmid was constructed by replacing the pUC ori in the pMON7124 plasmid with a second temperature-sensitive replicon control element.

7. The Bac-to-Bac baculovirus expression system according to claim 1, wherein The heterologous functional gene fragment expression frame in the donor plasmid is selected from the GFP expression frame.

8. A recombinant bacmid, characterized in that: The Bac-to-Bac baculovirus expression system according to claim 1 is prepared, specifically: the donor plasmid is transferred into DH10 Bac competent bacteria, blue-white screening is performed on a culture plate containing kanamycin, gentamicin, tetracycline, X-gal and IPTG, white colonies are picked, positive recombinant bacteria containing recombinant bacmid are obtained after identification, and the recombinant bacmid is extracted.

9. A recombinant baculovirus, characterized in that The recombinant bacmid according to claim 8 is transfected into insect cells for culture, and the recombinant baculovirus is harvested from the culture.

10. Use of the Bac-to-Bac baculovirus expression system according to claim 1, the recombinant bacmid according to claim 8 or the recombinant baculovirus according to claim 9 in preparing a heterologous protein.

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