Anchoring sequence recognized by T7 phage, DNA vaccine recombinant plasmid and application
阅读说明：本技术 一种t7噬菌体识别的锚定序列、dna疫苗重组质粒及应用 (Anchoring sequence recognized by T7 phage, DNA vaccine recombinant plasmid and application ) 是由 徐海 李玲 李睿婷 郭子杰 洪伟鸣 朱善元 于 2021-07-07 设计创作，主要内容包括：本发明属于基因工程技术领域,涉及一种T7噬菌体识别的锚定序列、DNA疫苗重组质粒及应用。本发明利用基因工程方法,调取T7噬菌体基因组左右两侧重复序列中的基因片段,并进行优化组合拼接成候选锚定序列,然后将其插入DNA疫苗质粒载体；建立荧光定量PCR检测方法,评价T7噬菌体对携带候选锚定序列的DNA疫苗载体的包裹效率,最终确定最佳锚定序列。应用T7噬菌体识别锚定序列并包裹DNA疫苗载体,并在细胞上验证T7噬菌体介导DNA疫苗载体转运、表达。本发明的携带锚定序列的DNA疫苗载体可独立于T7噬菌体进行基因的插入或替换,操作方便、运用广泛。(The invention belongs to the technical field of genetic engineering, and relates to an anchoring sequence recognized by a T7 bacteriophage, a DNA vaccine recombinant plasmid and application thereof. The invention uses a gene engineering method to call gene segments in repetitive sequences at the left and right sides of a T7 bacteriophage genome, and the gene segments are optimized, combined and spliced into candidate anchoring sequences, and then are inserted into a DNA vaccine plasmid vector; and (3) establishing a fluorescence quantitative PCR detection method, evaluating the wrapping efficiency of the T7 phage on the DNA vaccine vector carrying the candidate anchoring sequence, and finally determining the optimal anchoring sequence. T7 bacteriophage is used for recognizing the anchoring sequence and wrapping the DNA vaccine vector, and T7 bacteriophage is verified on cells to mediate the transportation and expression of the DNA vaccine vector. The DNA vaccine vector carrying the anchoring sequence can be independent of T7 bacteriophage to perform gene insertion or replacement, and has convenient operation and wide application.)
1. An anchor sequence recognized by a T7 phage, wherein the anchor sequence is set forth as SEQ ID No. 1.
2. A DNA vaccine recombinant plasmid having an anchor sequence recognized by a T7 bacteriophage, comprising a DNA vaccine vector and the anchor sequence recognized by the T7 bacteriophage of claim 1.
3. The DNA vaccine recombinant plasmid of claim 2, wherein the DNA vaccine vector is selected from pcDNA, pEGFP, pSV2, pBV, pJV, or pBJ.
4. The DNA vaccine recombinant plasmid of claim 3, wherein the DNA vaccine vector is pcDNA3.0.
5. A phage host strain into which the recombinant plasmid for DNA vaccine of any one of claims 2 to 4 has been introduced.
6. The phage host strain of claim 5, wherein the strain is E.coli BL 21.
7. A method for packaging and transporting a DNA vaccine vector, comprising: the anchoring sequence recognized by the T7 bacteriophage of claim 1 is inserted into a DNA vaccine vector, the eukaryotic expression vector is wrapped inside a capsid by recognizing the anchoring sequence in the T7 bacteriophage replication and assembly process, the complete T7 bacteriophage particle is used as a transport tool to realize the intracellular transport of the DNA vaccine vector, and the intracellular protein expression is carried out.
8. The method for T7 phage packaging and transporting DNA vaccine vectors of claim 7, wherein the target for T7 phage transportation is eukaryotic cells.
9. The anchoring sequence recognized by the T7 bacteriophage of claim 1, the DNA vaccine recombinant plasmid of any one of claims 2 to 4, the T7 bacteriophage of claim 7 or 8 for wrapping and transporting DNA vaccine vectors, and their use in DNA vaccine development.
Technical Field
The invention belongs to the technical field of genetic engineering, and relates to an anchoring sequence recognized by a T7 bacteriophage, a DNA vaccine recombinant plasmid containing the anchoring sequence, a bacteriophage host strain containing the DNA vaccine recombinant plasmid, a method for wrapping and transporting a DNA vaccine vector, and applications of the anchoring sequence and the DNA vaccine recombinant plasmid, in particular to a method for screening the anchoring sequence from a T7 bacteriophage genome and inserting the anchoring sequence into the DNA vaccine vector, verifying the wrapping capacity of the T7 bacteriophage on the DNA vaccine vector carrying the anchoring sequence, and enabling the bacteriophage to transport the DNA vaccine vector into cells to realize the expression of foreign genes.
Background
The DNA vaccine is a novel vaccine developed in the last 90 years, and is a eukaryotic expression vector containing an antigen encoding gene is introduced into cells, and an organism is induced to generate an immune response by expressing an antigen intracellularly. The immune pathway can not only stimulate humoral immune response, but also induce high-level cellular immunity, especially Cytotoxic T Lymphocyte (CTL) response, and has great advantages in the prevention and treatment of diseases. The DNA vaccine has wide prospect as the representative of the third-generation vaccine, but the DNA vaccine is easily digested by various enzymes in vivo and gradually degraded before entering cells, so that the quantity of the DNA vaccine effectively entering the cells is reduced, and the immune protection efficiency is not satisfactory. Although the immune efficiency of the DNA vaccine can be improved by strategies such as biomaterial wrapping, injection mode improvement and the like, the DNA vaccine is difficult to popularize due to the restriction of factors such as process, cost and the like, so that the innovative DNA vaccine transportation technology becomes a current and future research hotspot.
The T7 bacteriophage is a virulent bacteriophage infecting Escherichia coli, replicated and assembled in the cytoplasm of Escherichia coli, and its nucleic acid material is enveloped in a head capsid with a diameter of about 50 nm. The interior space of the capsid can be used to load exogenous DNA vaccine vectors as if a piece of armor had been worn over the otherwise naked DNA vaccine. T7 bacteriophage is used as a transfer carrier of DNA vaccine, and is easy to be recognized, phagocytized and degraded by immune cells, and the asymmetry of bacteriophage particles is beneficial to inducing T cell dependent immune response. The phage particles have strong stability and strong resistance to physical and chemical factors, and are stable in a body fluid environment, so that the DNA vaccine vectors wrapped inside are effectively protected, and the DNA vaccine vectors are efficiently transported to cells to realize antigen expression.
The information disclosed in this background is intended to enhance an understanding of the invention and should not be taken as an acknowledgement or any form of suggestion that the information forms prior art that is already known to a person skilled in the art.
Disclosure of Invention
The invention aims to solve the difficulties encountered in the process of transporting DNA vaccine vectors, and provides an anchoring sequence recognized by T7 bacteriophage, a DNA vaccine recombinant plasmid, T7 bacteriophage package, a method for transporting DNA vaccine vectors and application thereof.
In order to achieve the above object, the first aspect of the present invention provides an anchor sequence recognized by the T7 phage, the anchor sequence being represented by SEQ ID NO. 1. Specifically, the invention selects a recognition region in the T7 phage assembly process from the genome based on the mechanism of the T7 phage replication and assembly process, performs optimized combination, and finally determines an anchoring sequence as shown in SEQ ID NO. 1.
In a second aspect of the present invention, there is provided a DNA vaccine recombinant plasmid having an anchor sequence recognized by the T7 bacteriophage, comprising a DNA vaccine vector and the above-mentioned anchor sequence recognized by the T7 bacteriophage.
The DNA vaccine vector of the present invention is not particularly limited, and may be selected from pcDNA, pEGFP, pSV2, pBV, pJV or pBJ. Preferably, the DNA vaccine vector is pcdna3.0.
In a third aspect, the present invention provides a phage host strain into which the above-described recombinant plasmid for a DNA vaccine has been introduced. The strain may be escherichia coli BL 21.
According to a specific embodiment of the present invention, the anchoring sequence is inserted into a non-essential region of a DNA vaccine vector (eukaryotic expression vector) pcDNA3.0 using a molecular biological method, and the recombinant plasmid vector is introduced into a T7 bacteriophage Escherichia coli BL21 host. Infecting the colibacillus host carrying the DNA vaccine vector with T7 bacteriophage, and recovering filial generation T7 bacteriophage. Detecting the content of the DNA vaccine vector wrapped in the T7 bacteriophage by using the established fluorescent quantitative PCR method; the phage was inoculated into cultured DC cells and expression of the reporter gene (EGFP) in the cells was detected confocal.
In a fourth aspect, the present invention provides a method for packaging and transporting a DNA vaccine vector, the method comprising: the anchoring sequence recognized by the T7 phage is inserted into a DNA vaccine vector, the eukaryotic expression vector is wrapped inside the capsid by recognizing the anchoring sequence in the T7 phage replication and assembly process, the complete T7 phage particle is used as a transport tool to realize the transport of the DNA vaccine vector into cells, and protein expression is carried out in the cells.
According to the present invention, the target for T7 phage translocation is eukaryotic cells.
The anchoring sequence recognized by the T7 bacteriophage, the DNA vaccine recombinant plasmid and the method for wrapping and transporting the DNA vaccine vector by the T7 bacteriophage can be used for developing the DNA vaccine.
The invention uses a gene engineering method to call gene segments in repetitive sequences at the left and right sides of a T7 bacteriophage genome, and the gene segments are optimized, combined and spliced into candidate anchoring sequences, and then are inserted into a DNA vaccine plasmid vector; and (3) establishing a fluorescence quantitative PCR detection method, evaluating the wrapping efficiency of the T7 phage on the DNA vaccine vector carrying the candidate anchoring sequence, and finally determining the optimal anchoring sequence. T7 bacteriophage is used for recognizing the anchoring sequence and wrapping the DNA vaccine vector, and T7 bacteriophage is verified on cells to mediate the transportation and expression of the DNA vaccine vector.
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention screens the optimal anchoring sequence, can be efficiently identified by T7 bacteriophage and wraps more than 90 percent of DNA vaccine vectors.
(2) The T7 bacteriophage coated DNA vaccine carrier can effectively resist the degradation of nuclease, and reduce the risk of degradation of DNA vaccine in the transportation process.
(3) The DNA vaccine vector carrying the anchoring sequence can be independent of T7 bacteriophage to perform gene insertion or replacement, and has convenient operation and wide application.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Drawings
The above and other objects, features and advantages of the present invention will become more apparent by describing in more detail exemplary embodiments thereof with reference to the attached drawings.
FIG. 1 is a technical scheme of the T7 phage-packaged DNA vaccine vector of the present invention.
FIG. 2 shows the splicing of candidate anchor sequences and recombinant DNA vaccine vector construction.
FIG. 3 shows the results of the expression ability test of the DNA vaccine vector carrying the anchor sequence.
Figure 4 shows protection of T7 phage package against DNA vaccine vectors.
FIG. 5 shows a DNA vaccine vector fluorescent quantitative PCR method.
FIG. 6 shows a comparison of the efficiency of T7 phage wrapping different anchor sequences.
Figure 7 shows T7 phage transfer of DNA vaccine vectors into intracellular expression.
Detailed Description
Preferred embodiments of the present invention will be described in more detail below. While the following describes preferred embodiments of the present invention, it should be understood that the present invention may be embodied in various forms and should not be limited by the embodiments set forth herein.
Example 1 construction of recombinant DNA vaccine vector carrying candidate anchor sequence, the route of construction is shown in FIG. 1
1. PCR amplification of candidate anchor sequences
Using the T7 phage genome as a template, the primer F1/R1 amplifies the left 1 to 276 (fragment a) of the genome, as shown in lane 2 in FIG. 2-A; primer F2/R2 amplified right 39681 to 39936 (fragment b) as shown in lane 3 in FIG. 2-A; primer F3/R3 amplified the right side 38981 to position 39371 (fragment c), as shown in lane 1 in FIG. 2-A. As the 5 'end of the fragment a is complementary to the 3' end of the fragment B, the fragments a and B can be spliced into a complete sequence in a PCR system, primers F4/R4 (corresponding to the fragment d), F5/R5 (corresponding to the fragment e), F6/R6 (corresponding to the fragment F) and F7/R7 (corresponding to the fragment g) are designed by taking the complete sequence as a template, and fragments with the size of about 250bp in different regions of the template sequence are amplified by PCR, as shown in B of FIG. 2. Splicing the fragment c with the fragments d, e, f and g by using an SOE-PCR method respectively, wherein the operation steps are as follows: redesigning reverse primers R3d, R3e, R3f and R3g at the downstream of the fragment c, and introducing sequences of fragments d, e, f and g with the length of 20bp at the 5' end of the primers; and (3) carrying out secondary amplification by taking the fragment C AS a template and using primers F3/R3d, F3/R3e, F3/R3F and F3/R3g, recovering the amplified fragments, mixing the amplified fragments with fragments d, e, F and g respectively to serve AS templates, and carrying out SOE-PCR splicing on the primers F3/R4, F3/R5, F3/R6 and F3/R7 to obtain 4 candidate Anchor sequences (Anchor Sequence) AS1, AS2, AS3 and AS4, wherein the primers are shown AS C in FIG. 2. The primer sequences used are shown in Table 1.
The PCR amplification conditions were as follows:
the reaction procedure is as follows: 3min at 94 ℃; 30 cycles of 94 ℃ for 30sec, 54 ℃ for 30sec, 72 ℃ for 30 sec; 10min at 72 ℃.
SOE-PCR amplification conditions were as follows:
the reaction procedure is as follows: 94 ℃ for 30sec, 56 ℃ for 30sec, 72 ℃ for 5 min; sec 94 deg.C for 3 min; 30 cycles of 94 ℃ 30sec, 56 ℃ 30sec, 72 ℃ 45 sec; 10min at 72 ℃.
2. Construction of recombinant DNA vaccine vector carrying anchoring sequence
The amplified candidate anchoring sequence and DNA vaccine vector pcDNA3.0 are subjected to Bgl II and Nru I double enzyme digestion treatment, the gel recovery vector is connected with the fragment, and DH5 alpha is transformed and inserted into the eukaryotic expression vector pcDNA3.0 to construct a recombinant DNA vaccine vector containing the anchoring sequence. The eukaryotic expression vector pcDNA3.0 has the following double enzyme digestion conditions:
and (3) uniformly mixing the reaction system, placing the mixture in water bath at 37 ℃ for acting for 4 hours, carrying out 2% agarose gel electrophoresis identification on the enzyme digestion product, and then carrying out gel cutting recovery identification on the gel recovery kit.
Connecting the recovered anchoring sequence with a DNA vaccine vector, wherein the reaction system is as follows:
anchoring sequence 2.0 u L
pcDNA3 vector 3.0. mu.L
Ligation mix 5μL
The reaction system was mixed well and placed in a water bath at 16 ℃ for 1 hour to chemically transform E.coli DH 5. alpha. competent cells, and positive colonies were selected on ampicillin resistant plates. Extracting recombinant plasmid, and identifying the insertion condition of the anchoring sequence by BglII and NruI double enzyme digestion, as shown in D of figure 2, successfully inserting the target gene of about 650 bp.
3. Expression verification of recombinant DNA vaccine vector reporter gene
The anchor sequence-inserted DNA vaccine vector constructed in step 2 of example 1 was extracted in a large amount, and the content of the recombinant plasmid was determined by Nanodrop and adjusted to a concentration of 100 ng/. mu.l. Mu.l of recombinant DNA vaccine vector and 2. mu.l of TurboFect (Thermo) transfection reagent are added into 100. mu.l of serum-free DMEM culture solution and mixed evenly, and the mixture is acted for 15 minutes at room temperature. The mixture was added to 293T cells seeded in 24-well plates to a monolayer and gently mixed. And (5) continuing culturing in a carbon dioxide incubator, and detecting the surface condition of the reporter gene EGFP by a fluorescence microscope for 24 hours. As can be seen from FIG. 3, the insertion of the anchor sequence in the nonessential region of pcDNA3.0 did not affect the expression of the EGFP reporter gene at the multicloning site of the vector.
Example 2 protection of phage T7 packaging against DNA vaccine vectors
1. DNA vaccine vector-packaged T7 phage miniprep
The recombinant bacteria constructed in step 2 of example 1 were streaked on ampicillin-resistant plates. The single colony was inoculated with 3mL of LB medium containing ampicillin resistance and cultured overnight in a shaker at 37 ℃. Coli carrying a DNA vaccine vector (without anchor sequence inserted) was also cultured as a control. The next day, the two groups of seed solutions were transferred to 3mL of fresh culture medium at a ratio of 1:100, and the culture was continued until OD was reached600Each of 5 μ L T7 phage seeds (10) was inoculated at nm 1.010pfu/mL), culture was continued for 2-3 hours until the E.coli was completely lysed. At the moment, T7 bacteriophage infects recombinant bacteria carrying anchoring sequence, and the inside of capsid is wrapped with DNA vaccine vector; infecting escherichia coli without anchor sequence, and not wrapping the DNA vaccine vector inside the capsid, as shown in fig. 4B, E.
2. PCR verification of protection of T7 phage capsid encapsulation against DNA vaccine vectors
The small amount of T7 phage prepared in the above step was centrifuged for 10 minutes at 5000 rpm to remove E.coli debris. And (3) adding RNase A and DNase I with the final concentration of 1 mu L/mL into 500 mu L of supernatant, digesting for 2 hours at 37 ℃, removing the Escherichia coli genome and free DNA vaccine vector in the system, extracting nuclease by using equal volume of chloroform, and stopping the digestion reaction. And continuously diluting the digestion product by 10 times, taking 1 mu L as a template, and detecting the reporter gene EGFP in the DNA vaccine vector by utilizing PCR. The PCR amplification conditions for EGFP reporter gene were as follows:
the reaction procedure is as follows: 3min at 94 ℃; 30 cycles of 94 ℃ for 30sec, 54 ℃ for 30sec, 72 ℃ for 30 sec; 10min at 72 ℃.
As can be seen from C, F of FIG. 4, the DNA vaccine vector without the anchor sequence inserted could not be packaged by the T7 phage, the vector was free from the phage, the reporter gene could be detected by PCR before nuclease digestion treatment and could not be detected after digestion treatment (C of FIG. 4). In contrast, the DNA vaccine vector inserted with the anchor sequence could be wrapped with T7 phage, and nuclease could not damage the DNA vaccine vector due to protection of phage capsid, and reporter gene could be detected both before and after digestion (F of fig. 4).
Example 3 evaluation of efficiency of T7 phage-coated DNA vaccine vectors
1. Establishment of DNA vaccine carrier fluorescent quantitative PCR method
A fluorescent quantitative PCR method is established by detecting a reporter gene EGFP by using the recombinant DNA vaccine vector prepared in the step 2 of the embodiment 1 as a template. Firstly, the recombinant DNA vaccine vector (plasmid) is quantified by using Nanodrop, and the corresponding relation between the copy number and the mass of the plasmid is calculated according to the molecular weight of the recombinant DNA vaccine vector. Adjusting the concentration of the DNA vaccine carrier, respectively diluting the DNA vaccine carrier to 1, 10, 100, 1000, 10000, 100000 and 1000000 copies/mu L in a gradient manner, taking 1 mu L of carrier diluent as a template, establishing a fluorescence quantitative PCR detection method, drawing a standard curve as shown in C of figure 5, obtaining a calculation formula y which is-3.257 x +38.45, and using the calculation formula to calculate the copy number of the DNA vaccine carrier in the sample to be detected.
2. Effect of different Anchor sequences on the efficiency of T7 phage encapsidation
The anchor sequences AS1, AS2, AS3 and AS4 gene fragments obtained in step 1 of example 1 were inserted into DNA vaccine vectors respectively according to the technical approach in step 2 of example 1, to construct 4 recombinant DNA vaccine vectors. The T7 phages infected E.coli carrying different recombinant DNA vaccine vectors, respectively, and samples of T7 phage-coated recombinant DNA vaccine vectors were miniprepped according to the method of step 1 of example 2, and subjected to nuclease digestion in the method of step 2 of example 2, while setting undigested controls. According to the fluorescent quantitative PCR method established in step 1 of example 3, the copy numbers of the recombinant DNA vaccine vectors (plasmids) before and after digestion of the sample to be tested were determined, respectively. According to the formula: the encapsulation efficiency (number of plasmid copies after digestion/plasmid copies before digestion) × 100%, the effect of different anchor sequences on encapsulation was calculated. As can be seen from A in FIG. 6, the DNA vaccine vector carrying the anchor sequence AS2 can be efficiently packaged by the T7 phage with a packaging efficiency AS high AS 95% (B in FIG. 6), which is the optimal anchor sequence, and the gene sequence is shown in SEQ ID NO. 1.
Example 4T7 phage transfer of DNA vaccine vectors into intracellular expression
1. Large-scale preparation and purification of T7 bacteriophage wrapping DNA vaccine vector
Recombinant bacteria carrying the AS2 anchor sequence DNA vaccine vector were streaked in four regions on ampicillin resistant plates. The single colony was inoculated with 5mL of LB medium containing ampicillin resistance and cultured overnight in a shaker at 37 ℃. Coli carrying a DNA vaccine vector (without anchor sequence inserted) was also cultured as a control. The next day, the two groups of seed solutions were transferred to 500mL of fresh culture medium at a ratio of 1:100, and the culture was continued until OD reached600Each of 500 μ L T7 phage seeds (10) was inoculated at nm 1.010pfu/mL), culture was continued for 2-3 hours until the E.coli was completely lysed. RNase A and DNase I were added to the culture system at a final concentration of 1. mu.L/mL, and the culture was continued for 30 minutes at 37 ℃ with a shaker. Centrifuging for 15 min at 5000 r, recovering supernatant, adding 10% PEG-8000 and 3M NaCl, mixing, and standing at 4 deg.C overnight. The next day, 12000 rpm, 15 min centrifugation, recovery of T7 phage pellet, 5mL SM buffer heavy suspension. Adding 1% Trition-114 solution into the resuspension solution, mixing thoroughly, acting at 4 deg.C for 30 min, then acting in 37 deg.C water bath for 10min, centrifuging at 8000 rpm for 10min, recovering supernatant, and repeating the above steps once. The titer of the recovered T7 phage is determined by a double-layer agar sandwich method, and the concentration is adjusted to 1011pfu/mL, and freezing for later use.
2. T7 bacteriophage transports DNA vaccine vectors into intracellular expression
Preparing SPF chicken bone marrow-derived dendritic cells by Inaba method, subjecting the dendritic cells cultured by induction to day 6 to trypsinization, and adjusting cell density to 10 × 106one/mL. Cell slide was plated on a 24-well cell plate, 1mL of dendritic cells was inoculated uniformly, and the cells were cultured overnight in a carbon dioxide incubator at 37 ℃. The next day, 50 μ L of T7 phage frozen in step 1 of example 4 was inoculated and incubated with dendritic cells for 1 hour, and the culture medium in the wells was aspirated and washed gently once with PBS buffer. 1mL of 10% FBS in DMEM was added to each well and incubation was continued for 36 hours. Cell slide was removed and washed 1 time with PBS. Cells on the slide were fixed with 1% cell fixative for 15 min at room temperature and washed 2 times with PBS. Adding a drop of mounting solution containing DAPI on the glass slide, covering the cell surface on the slide to the mounting solution, and observing the expression condition of the reporter gene EGFP on the DNA vaccine carrier wrapped by the T7 bacteriophage by using a laser confocal microscope. As can be seen in FIG. 7, the T7 phage-coated DNA vaccine vector can enter dendritic cells and achieve intracellular protein expression.
In conclusion, the optimal anchoring sequence is screened and used for inserting the DNA vaccine vector, the recombinant vector is constructed and introduced into escherichia coli, the T7 phage can identify the recombinant DNA vaccine vector when being replicated and assembled in the escherichia coli, and the recombinant DNA vaccine vector is wrapped in the capsid, so that the DNA vaccine vector is protected from enzyme damage, and the protein expression of the DNA vaccine vector in cells is realized by utilizing a transport tool for finishing the action of T7 phage particles. The invention builds a good platform for the research and development of DNA vaccines.
Having described embodiments of the present invention, the foregoing description is intended to be exemplary, not exhaustive, and not limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments.
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