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Anchor sequence identified by T7 phage, DNA vaccine recombinant plasmid and application

A DNA vaccine and anchor sequence technology, applied in the field of genetic engineering technology, can solve problems such as difficulty in popularization, unsatisfactory immune protection efficiency, and sudden reduction in the number of DNA vaccines, so as to reduce the risk of degradation and facilitate operation , using a wide range of effects

Active Publication Date: 2021-11-05
JIANGSU AGRI ANIMAL HUSBANDRY VOCATIONAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As a representative of the third-generation vaccine, DNA vaccine has broad prospects. However, before entering cells, DNA vaccines are susceptible to digestion by various enzymes in the body and are gradually degraded, resulting in a sharp decrease in the number of DNA vaccines that can effectively enter cells, and immune protection not efficient
Although strategies such as encapsulation of biological materials and improved injection methods can be used to improve the immune efficiency of DNA vaccines, it is difficult to promote them due to factors such as technology and cost. Therefore, innovative DNA vaccine transfer technology has become a current and future research hotspot.

Method used

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  • Anchor sequence identified by T7 phage, DNA vaccine recombinant plasmid and application
  • Anchor sequence identified by T7 phage, DNA vaccine recombinant plasmid and application
  • Anchor sequence identified by T7 phage, DNA vaccine recombinant plasmid and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Construction of a recombinant DNA vaccine vector carrying a candidate anchor sequence, the construction route method is as follows figure 1 shown

[0030] 1. PCR amplification of candidate anchor sequences

[0031] Using the T7 phage genome as a template, primer F1 / R1 amplifies positions 1 to 276 on the left side of the genome (fragment a), such as figure 2 -Shown in 2 swimming lanes in A; primer F2 / R2 amplifies positions 39681 to 39936 on the right (fragment b), as figure 2 -Shown in 3 swimming lanes in A; Primer F3 / R3 amplifies position 38981 to 39371 on the right side (fragment c), as figure 2 -Shown in lane 1 in A. Since the 5' end of fragment a is complementary to the 3' end of fragment b, fragments a and b can be spliced ​​into a complete sequence in the PCR system. Using the complete sequence as a template, design primers F4 / R4 (corresponding to fragment d), F5 / R5 (corresponding to fragment e), F6 / R6 (corresponding to fragment f) and F7 / R7 (corre...

Embodiment 2T7

[0052] The protection of embodiment 2T7 phage encapsulation to DNA vaccine carrier

[0053] 1. Small-scale preparation of T7 phage encapsulating DNA vaccine vector

[0054] The recombinant bacteria constructed in Step 2 of Example 1 were cultured in four sections on the ampicillin-resistant plate. Pick a monoclonal colony and inoculate 3 mL of LB culture medium containing ampicillin resistance, and culture overnight on a shaker at 37°C. At the same time, Escherichia coli carrying the DNA vaccine vector (without inserting the anchor sequence) was cultured as a control. On the next day, transfer the cultured two groups of seed solutions to 3 mL of fresh culture solution at a ratio of 1:100, and continue to cultivate to OD 600 nm=1.0, respectively inoculate 5 μL T7 phage seeds (10 10 pfu / mL), continue to cultivate for 2-3 hours until E. coli is completely lysed. At this time, the T7 phage infects the recombinant bacteria carrying the anchor sequence, and the inside of the cap...

Embodiment 3T7

[0060] Efficiency evaluation of embodiment 3T7 phage encapsulating DNA vaccine carrier

[0061] 1. Establishment of DNA vaccine vector fluorescent quantitative PCR method

[0062] Using the recombinant DNA vaccine vector prepared in Step 2 of Example 1 as a template, a fluorescent quantitative PCR method was established by detecting the reporter gene EGFP. First, use Nanodrop to quantify the recombinant DNA vaccine vector (plasmid), and calculate the corresponding relationship between plasmid copy number and quality according to the molecular weight of the recombinant DNA vaccine vector. Adjust the concentration of the DNA vaccine carrier, serially dilute to 1, 10, 100, 1000, 10000, 100000 and 1000000 copies / μL respectively, take 1 μL of the carrier dilution as a template, establish a fluorescent quantitative PCR detection method, and draw a standard curve such as Figure 5 As shown in C, the calculation formula y=-3.257x+38.45 is obtained, which is used to calculate the copy...

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Abstract

The invention belongs to the technical field of genetic engineering, and relates to an anchoring sequence identified by a T7 phage, a DNA vaccine recombinant plasmid and application. Gene segments in repetitive sequences on the left side and the right side of a T7 phage genome are retrieved by using a gene engineering method and are optimally combined and spliced into candidate anchoring sequences, and then the candidate anchoring sequences are inserted into a DNA vaccine plasmid vector; and a fluorescent quantitative PCR detection method is established, the wrapping efficiency of the T7 phage on the DNA vaccine vector carrying the candidate anchoring sequences are evaluated, and finally the optimal anchoring sequence is determined. The T7 phage is used for identifying the anchoring sequence and wrapping the DNA vaccine vector, and the transport and expression of the T7 phage mediated DNA vaccine vector are verified on cells. The DNA vaccine vector carrying the anchoring sequence can be used for gene insertion or replacement independent of the T7 phage, and is convenient to operate and wide in application.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to an anchor sequence recognized by T7 phage, a DNA vaccine recombinant plasmid containing the anchor sequence, a phage host strain containing the DNA vaccine recombinant plasmid, and a DNA vaccine vector for encapsulation and transfer The method, and their application, specifically relate to screening the anchor sequence from the T7 phage genome and inserting the DNA vaccine vector, verifying the ability of the T7 phage to wrap the DNA vaccine vector carrying the anchor sequence, and translating the DNA vaccine vector into the cell to achieve exogenous gene expression. Background technique [0002] DNA vaccine is a new type of vaccine developed in the 1990s. It refers to the introduction of eukaryotic expression vectors containing antigen-encoding genes into cells, and the expression of antigens in cells induces an immune response in the body. This immune pathway can not...

Claims

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Application Information

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IPC IPC(8): C12N15/34C12N15/85C12N15/70C12N15/65C12N1/21C12R1/19
CPCC07K14/005C12N15/85C12N15/70C12N15/65C12N2795/10222C12N2795/10242Y02A50/30
Inventor 徐海李玲李睿婷郭子杰洪伟鸣朱善元
Owner JIANGSU AGRI ANIMAL HUSBANDRY VOCATIONAL COLLEGE
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