CN109053894B - Construction method and function verification method of recombinant attenuated salmonella typhimurium - Google Patents
Construction method and function verification method of recombinant attenuated salmonella typhimurium Download PDFInfo
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Abstract
The invention discloses a method for constructing and functionally verifying a GrB recombinant single-chain antibody, which comprises the following steps: (1) constructing pcDNA3.3c-GrB-Gala recombinant plasmid; (2) constructing a pcDNA3.3c-GrB-Gala attenuated Salmonella typhimurium VNP20009 recombinant strain; (3) the GrB recombinant plasmid realizes the eukaryotic expression of protein GrB after invading cells by the characteristics of attenuated salmonella typhimurium VNP20009 intracellular invasion, and verifies the killing effect of the protein on the cells. The invention adopts the characteristic of intracellular invasion of the attenuated salmonella typhimurium VNP20009 to realize the expression of the human-derived toxin protein, and simultaneously, a large number of researches show that the attenuated salmonella typhimurium VNP20009 can be specifically gathered in solid tumors, can realize the purpose of specifically killing tumor cells, and provides experimental basis for tumor treatment research.
Description
Technical Field
The invention belongs to the fields of biological pharmacy and tumor medicine, and mainly relates to a method for constructing and functionally verifying a GrB recombinant single-chain antibody.
Background
Granzyme (Gr) is a generic name for a family of serine proteases in the granule of killer T lymphocytes and Natural Killer (NK) cells. Human granzymes share A, B, C types, and GrB is initially stored synthetically as a zymogen in CTL particles. When CTL recognizes the target cell, the N-terminal acid dipeptide of GrB is cleaved to become an active form. The active GrB can enter target cells through perforin or receptors, and cut various structural and functional proteins containing Asp-x and Gln-x sites to cause apoptosis.
The attenuated salmonella typhimurium VNP20009 is used as a treatment vector, the characteristics of intracellular invasion and specific aggregation of the attenuated salmonella typhimurium in solid tumors are comprehensively applied, and the tumor killing specificity is further expanded.
Nowadays, a novel therapeutic Drug of Antibody Drug Conjugates (ADCs) is provided for the clinical treatment of cancer, and the invention provides a certain experimental data basis for the development of ADC Drug treatment by applying the concept and combining with the early-stage basis of an experiment.
Disclosure of Invention
The invention aims to provide an attenuated salmonella typhimurium-mediated plasmid transfection technology in a eukaryotic plasmid expression process.
The second purpose of the invention is to successfully construct a recombinant attenuated salmonella typhimurium pcDNA3.3c-GrBGala-VNP20009, which has the effect of specifically killing tumor cells.
The construction method of the GrB recombinant single-chain antibody comprises the following steps:
(1) obtaining a GrB-Gala fragment: taking pET302-GrB-Gala as a template and SEQ ID No.3 and SEQ ID No.4 sequences as primers, and carrying out PCR amplification to obtain a GrB-Gala fragment shown in SEQ ID No. 1;
(2) constructing pcDNA3.3c-GrB-Gala recombinant plasmid: connecting the GrB-Gala fragment obtained in the step (1) to pcDNA3.3c by a method of enzyme digestion and enzyme ligation, wherein the sequence of the GrB-Gala fragment is as follows: in the plasmid of SEQ ID No.2, the recombinant plasmid was named pcDNA3.3c-GrB-Gala;
(3) constructing pcDNA3.3c-GrB-Gala recombinant strain: transferring the pcDNA3.3c-GrB-Gala recombinant plasmid in the step (1) into E.coli Top10 to obtain a recombinant strain;
(4) constructing pcDNA3.3c-GrB-Gala recombinant attenuated Salmonella typhimurium VNP 20009: obtaining a recombinant plasmid from the recombinant strain obtained in the step (3), and then placing the recombinant plasmid into Salmonella typhimurium VNP20009 by an electrotransformation method, wherein the electrotransformation conditions are as follows: 1800V, 25uF, 200 omega and 4.7ms, and the recombined salmonella typhimurium is named pcDNA3.3c-GrBGAla-Gala-VNP 20009.
The recombinant attenuated salmonella typhimurium pcDNA3.3c-GrB-Gala-VNP20009 constructed by the method.
The constructed antibody function verification method comprises the following steps:
(1) and (3) carrying out mixed culture on the recombinant attenuated salmonella typhimurium VNP20009 obtained in the step 1 and B16F10 cells for 2 hours according to the ratio of the cells to the bacteria of 100:1, and then replacing a fresh culture medium containing the streptomycin to continuously culture for 24 hours.
(2) And (2) respectively carrying out photographing observation on the cells cultured in the step (1) and calculating the specific killing effect of the protein on the cells by adopting an MTT colorimetric method.
The steps adopt the technology of eukaryotic plasmid transfection and expression.
The invention has the beneficial technical effects that:
(1) the attenuated salmonella typhimurium VNP20009 is adopted to mediate eukaryotic plasmid transfected cells to realize eukaryotic expression, and experimental basis is provided for subsequent bacterial treatment means;
(2) the invention utilizes the targeting specificity of a single-chain antibody and the specific aggregation property of attenuated salmonella typhimurium VNP20009 in solid tumors, combines the cell killing effect of human toxin GrB, belongs to a novel therapeutic drug of an antibody drug conjugate, and can better realize the effect of specifically killing cells.
Drawings
FIG. 1 shows the results of GrB-Gala PCR validation; wherein a: Marker-DL2000, b: pcDNA3.3c-GrB-Gala plasmid, c: a GrB-Gala fragment;
FIG. 2 is a microscopic representation of the killing capacity of GrB protein on cells;
FIG. 3 shows the MTT assay result of GrB protein on cell killing ability.
Detailed Description
The invention will be further illustrated by the following examples.
The original plasmids of the invention are all from Biovector NCTT company.
Coli Top10 and attenuated Salmonella typhimurium VNP20009 of the present invention were purchased from Biovector NCTT.
All restriction enzymes used were purchased from NEB.
The T4DNA ligase used was purchased from Takara.
The DNA ligases used were all purchased from Takara.
Both LB solid medium and liquid medium were purchased from Solebao.
Example 1
PCR amplification is carried out by taking pET302-GrB-Gala as a template and SEQ ID No.3 and SEQ ID No.4 as primers to obtain a GrB-Gala fragment shown in SEQ ID No. 1.
Example 2
The obtained GrB-Gala fragment and plasmid pcDNA3.3c were subjected to double digestion with Age I and Bsiw I, the digestion products were purified and ligated into pcDNA3.3c (SEQ ID No.2) plasmid using T4 ligase, and the recombinant plasmid was named pcDNA3.3c-GrB-Gala. See the attached figure 1 of the specification.
Example 3
The attenuated salmonella typhimurium VNP20009 preserved at-80 ℃ is streaked and inoculated to an LB plate without resistance and cultured overnight at 37 ℃; picking a single colony in 5ml LB, and carrying out shaking culture at 37 ℃ for 12 h; according to the following steps: inoculating 100 proportion into 100ml LB to shake culture till bacterial OD is about 0.4; after ice-bath for 20min, centrifuging at 4 ℃ and 3000rpm for 10 min; washing thallus precipitate twice with 1/10 volume of precooled sterile deionized water, and centrifuging at 4 ℃ and 3000rpm for 10 min; washing thallus with 1/100 volume precooled 10% glycerol, centrifuging at 4 deg.C 3000rpm for 10 min; the pellet was resuspended in 1/100 volumes of pre-cooled 10% glycerol to make VNP20009 competent aliquots and retained at-80 ℃ for use.
Example 4
The obtained recombinant plasmid pcDNA3.3c-GrB-Gala is electrically transferred into an attenuated salmonella typhimurium VNP20009 by adopting a 0.1cm electric transfer cup, and the conditions are set as follows: 1.8kv, 200 omega, 25uF, 4.7ms of electric conversion; screening out positive clones through ampicillin resistance to obtain recombinant attenuated salmonella typhimurium VNP20009 which is named pcDNA3.3c-GrBGala-VNP 2000.
Example 5
B16F10 cells were seeded in 96-well plates (using penicillin and streptomycin free medium) at 10000/well and recombinant attenuated salmonella typhimurium VNP20009 was seeded at B16F10 cells: recombinant bacteria ═ 100:1 in the total volume of 200 mu L, culturing for 2h, changing to a culture medium containing penicillin and streptomycin, culturing for 24h, and detecting the specific killing effect of the protein on cells by adopting an MTT colorimetric method.
Sequence listing
<110> university of Nanchang
<120> construction method and function verification method of recombinant attenuated salmonella typhimurium
<130> 2018
<160> 4
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atcatcgggg gacatgaggc caagccccac tcccgcccct acatggctta tcttatgatc 60
tgggatcaga agtctctgaa gaggtgcggt ggcttcctga tacaagacga cttcgtgctg 120
acagctgctc actgttgggg aagctccata aatgtcacct tgggggccca caatatcaaa 180
gaacaggagc cgacccagca gtttatccct gtgaaaagac ccatccccca tccagcctat 240
aatcctaaga acttctccaa cgacatcatg ctactgcagc tggagagaaa ggccaagcgg 300
accagagctg tgcagcccct caggctacct agcaacaagg cccaggtgaa gccagggcag 360
acatgcagtg tggccggctg ggggcagacg gcccccctgg gaaaacactc acacacacta 420
caagaggtga agatgacagt gcaggaagat cgaaagtgcg aatctgactt acgccattat 480
tacgacagta ccattgagtt gtgcgtgggg gacccagaga ttaaaaagac ttcctttaag 540
ggggactctg gaggccctct tgtgtgtaac aaggtggccc agggcattgt ctcctatgga 600
cgaaacaatg gcatgcctcc acgagcctgc accaaagtct caagctttgt acactggata 660
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gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta 60
ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag 120
ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc 180
ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga 240
cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat 300
atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc 360
cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct 420
attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca 480
cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat 540
caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg 600
cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca gatcgcctgg 660
agacgccatc cacgctgttt tgacctccat agaagacacc gggaccgatc cagcctccgg 720
actctagagg atcgaaccct tactagtgcc accatggagt ttgggctgag ctgggtcttc 780
ctggtggcta tcttgaaggg tgtccagtgt gaggtggcta gcgaattcaa gcttcccggg 840
gtcgacctgc aggagctcct cgagagatct ggatcccacc atcaccacca tcaccaccac 900
gggtgagcgg ccgcttcatg aagggttcga tccctaccgg ttagtaatga gtttaaacgg 960
gggaggctaa ctgaaacacg gaaggagaca ataccggaag gaacccgcgc tatgacggca 1020
ataaaaagac agaataaaac gcacgggtgt tgggtcgttt gttcataaac gcggggttcg 1080
gtcccagggc tggcactctg tcgatacccc accgagaccc cattggggcc aatacgcccg 1140
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ctgagctggg tcttcctggt ggctatcttg 30
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Claims (2)
1. The construction method of the recombinant attenuated salmonella typhimurium pcDNA3.3c-GrB-Gala-VNP20009 is characterized by comprising the following steps:
(1) obtaining a GrB-Gala fragment: taking pET302-GrB-Gala as a template and SEQ ID No.3 and SEQ ID No.4 sequences as primers, and carrying out PCR amplification to obtain a GrB-Gala fragment shown in SEQ ID No. 1;
(2) constructing pcDNA3.3c-GrB-Gala recombinant plasmid: connecting the GrB-Gala fragment obtained in the step (1) to pcDNA3.3c by a method of enzyme digestion and enzyme ligation, wherein the sequence of the GrB-Gala fragment is as follows: in the plasmid of SEQ ID No.2, the recombinant plasmid was named pcDNA3.3c-GrB-Gala;
(3) constructing pcDNA3.3c-GrB-Gala recombinant strain: transferring the pcDNA3.3c-GrB-Gala recombinant plasmid in the step (2) into E.coli Top10 to obtain a recombinant strain;
(4) constructing pcDNA3.3c-GrB-Gala recombinant attenuated Salmonella typhimurium VNP 20009: obtaining a recombinant plasmid from the recombinant strain obtained in the step (3), and then placing the recombinant plasmid into Salmonella typhimurium VNP20009 by an electrotransformation method, wherein the electrotransformation conditions are as follows: 1800V, 25uF, 200 omega and 4.7ms, and the recombined salmonella typhimurium is named pcDNA3.3c-GrB-Gala-VNP 20009.
2. The functional verification method of the recombinant attenuated salmonella typhimurium pcDNA3.3c-GrB-Gala-VNP20009 is characterized by comprising the following steps:
(1) the recombinant attenuated salmonella typhimurium pcDNA3.3c-GrB-Gala-VNP20009 of claim 1 is mixed with B16F10 cells for 2 hours according to the ratio of the cells to the bacteria 100:1, and then fresh culture medium containing the streptomycin is replaced for further culture for 24 hours;
(2) and (2) respectively carrying out photographing observation on the cells cultured in the step (1) and calculating the specific killing effect of the recombinant attenuated salmonella typhimurium pcDNA3.3c-GrB-Gala-VNP20009 on the cells by adopting an MTT colorimetric method.
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