CN108913690B - PD-1 specific interference sequence, plasmid, attenuated salmonella and application in tumor resistance - Google Patents

Abstract

The invention belongs to the technical field of biology, and particularly relates to a PD-1 specific interference sequence, a plasmid, attenuated salmonella and application in tumor resistance. The PD-1 specific interference sequence is sequence 1, sequence 2 or sequence 3; wherein, the sense strand of the sequence 1 is shown as SEQ ID NO.1, the antisense strand is shown as SEQ ID NO.2, the sense strand of the sequence 2 is shown as SEQ ID NO.3, the antisense strand is shown as SEQ ID NO.4, the sense strand of the sequence 3 is shown as SEQ ID NO.5, and the antisense strand is shown as SEQ ID NO. 6. The PD-1 specific interference plasmid is prepared by connecting a PD-1 specific interference sequence into pGCSilencer neo2.0-U6 plasmid. The constructed attenuated salmonella carrier PD-1 specific interference plasmid is used for treating tumors, so that a new strategy and a preparation are provided for treating the tumors clinically, and great market economic benefits are generated.

Description

PD-1 specific interference sequence, plasmid, attenuated salmonella and application in tumor resistance

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a PD-1 specific interference sequence, a plasmid, attenuated salmonella and application in tumor resistance.

Background

According to the report of the registration annual report of Chinese tumors, 312 million new cases are reported each year, the death rate is nearly 200 million, and the Chinese cancer death rate is increased by 80 percent in the last 30 years, so that the new cases become the first cause of death of Chinese urban residents and the second cause of death of rural residents. Statistics in 2014 in the united states show that prostate cancer and lung cancer respectively exist in the first two male morbidity sites and breast cancer and lung cancer respectively exist in the first two female morbidity sites worldwide. With the change of modern environment, particularly the increase of haze, the incidence rate of lung cancer shows a remarkable rising trend. Cancer is one of the most important diseases which endanger the health of Chinese residents, and the economic burden and the adverse effect on the social development are increasingly highlighted. China now has medical expenses for cancer treatment as high as hundreds of billions of yuan every year and shows an increasing trend year by year. However, even if a lot of money is spent, patients with middle and advanced stage tumors are still difficult to cure.

PD-1/PD-L has made a major advance in the treatment of tumors at tumor immune checkpoints, and in 1992, the hypothesis of the presence of "key molecules for immune escape" in the tumor microenvironment was presented. PD-1(programmed death 1) programmed death receptor 1, an important immunosuppressive molecule. PD-1 is mainly expressed in activated T cells and B cells, and tumor cells will highly express the ligand of PD-1: PD-L1 and PD-L2, receptor and ligand binding results in inhibition of immune cell activation. The tumor microenvironment can induce infiltrated T cells to highly express PD-1 molecules, so that PD-1 channels in the tumor microenvironment are continuously activated, the functions of the T cells are inhibited, and the tumor cells cannot be killed. The anti-human PD-1 monoclonal antibody can block the pathway and restore the function of T cells, so that the cells can continuously kill tumor cells. In the years 1999 to 2002, the invention uses the antibody to block PD-1/PD-L1 combination so as to enhance immune response, and animal experiments prove that the antibody can successfully treat tumors. These findings provide a new therapeutic method for the current anti-tumor immunity escape, thereby laying the theoretical basis for the monoclonal antibody to treat tumors. Antibody therapy (anti-PD-1 therapy for short) designed based on this principle to block the binding of PD-1 and PD-L1 has been used in thousands of cancer patients, and has been confirmed to have significant efficacy in more than ten advanced cancers, including lung cancer, kidney cancer, melanoma, head and neck cancer, bladder cancer, breast cancer, liver cancer, stomach cancer, esophageal cancer, brain glioma, colon cancer, hodgkin's lymphoma, etc., and PD-1 therapy is currently the most effective immunotherapy.

Currently, inhibition of the PD-1 pathway has become a very important immune checkpoint in tumor immunotherapy. The use of monoclonal antibodies to inhibit the PD-1 pathway is effective in treating a variety of tumors including melanoma, but it is known that inhibition of signaling pathways can directly interfere with the expression of its genes in addition to the use of monoclonal antibodies. RNAi has become an important core topic in the fields of genetics and gene therapy. The method specifically degrades target RNA (mRNA) by double-stranded small interfering RNA with the length of 21-25nt in a base complementary pairing mode, thereby eliminating the function of the target RNA, blocking the regulation and control function of the target RNA or blocking the synthesis of corresponding protein of the target RNA. The discovery of RNA interference phenomenon greatly stimulates the research heat of nucleic acid biopharmaceutical technology. However, at present, no small interfering RNA sequences that inhibit the PD-1 pathway have been discovered.

Disclosure of Invention

The invention provides a PD-1 specific interference sequence, a plasmid, attenuated salmonella and application in tumor resistance, and develops a small interference RNA sequence capable of inhibiting a PD-1 pathway.

The invention provides a PD-1 specific interference sequence, wherein the PD-1 specific interference sequence is a sequence 1, a sequence 2 or a sequence 3; wherein, the sense strand of the sequence 1 is shown as SEQ ID NO.1, the antisense strand is shown as SEQ ID NO.2, the sense strand of the sequence 2 is shown as SEQ ID NO.3, the antisense strand is shown as SEQ ID NO.4, the sense strand of the sequence 3 is shown as SEQ ID NO.5, and the antisense strand is shown as SEQ ID NO. 6.

Preferably, the synthetic siRNA strand is designed before the PD-1 specific interference sequence is synthesized, and the design principle is as follows:

(1) selecting siRNA chains after 100 bases of PD-1 gene promoter;

(2) a siRNA strand of 5 '-AA (N19) TT-3' selected from the PD-1 gene is used as an siRNA sense strand; the siRNA antisense strand sequence is a complementary sequence of a sense strand of the siRNA;

(3) if 5 '-AA (N19) TT-3' is not found, then complement with 5 '-AA (N21) -3';

(4) if 5 '-AA (N21) -3' is not found, then complement with 5 '-NA (N21) -3'; (1) n in (1) to (3) may be any base, N19 represents 19 bases, and N21 represents 21 bases;

(5) in the selected siRNA strands, the GC ratio is 35-55%;

(6) if the siRNA chains meeting the requirements of (1) to (5) cannot be found out, the GC ratio is relaxed to 30 to 70 percent until the siRNA chains meeting the requirements of (1) to (5) are selected;

after the siRNA chains are selected, BamHI and HindIII sequences are respectively added at the two ends of each siRNA chain, and then PD-1 specific interference sequences are synthesized.

The invention also provides a PD-1 specific interference plasmid carrying the PD-1 specific interference sequence of claim 1, wherein the PD-1 specific interference plasmid is prepared by connecting the PD-1 specific interference sequence into pGCSilencer neo2.0-U6 plasmid.

Preferably, in the PD-1 specific interference plasmid carrying the PD-1 specific interference sequence of claim 1, the PD-1 specific interference plasmid is prepared by the following method:

s1, synthesizing a PD-1 specific interference sequence:

the PD-1 specific interference sequence is sequence 1, sequence 2 or sequence 3; wherein, the sense strand of the sequence 1 is shown as SEQ ID NO.1, the antisense strand is shown as SEQ ID NO.2, the sense strand of the sequence 2 is shown as SEQ ID NO.3, the antisense strand is shown as SEQ ID NO.4, the sense strand of the sequence 3 is shown as SEQ ID NO.5, and the antisense strand is shown as SEQ ID NO. 6;

s2, preparing a PD-1 specific interference plasmid;

s2 step A sequence 1 is taken as an example to illustrate the preparation method of the PD-1 specific interference plasmid,

s21, preparing a sense strand diluent and an antisense strand diluent;

s22, annealing the sense strand diluent and the antisense strand diluent to prepare PD1-siRNA double chains;

s23, linearization of plasmid vector and construction of PD-1 specific interference plasmid:

after the pGCSilencer neo2.0-U6 plasmid is subjected to double enzyme digestion by Bam H I and Hand III, a linearized plasmid DNA fragment is recovered by agarose gel, and a PD1-siRNA-1 double strand obtained from S22 is connected to the linearized plasmid DNA fragment to obtain a PD-1 specific interference plasmid pH1 Si-PD-1;

the method for preparing the PD-1 specific interference plasmid by using the sequence 2 and the sequence 3 is the same as the method for preparing the PD-1 specific interference plasmid by using the sequence 1, and the difference is that the nucleotide sequences of a sense strand and an antisense strand of the sequence 1, the sequence 2 and the sequence 3 are different.

Preferably, the present invention also provides an attenuated salmonella carrying a PD-1 specific interfering plasmid as described.

Preferably, the invention also provides an application of the PD-1 specific interference sequence in resisting melanoma.

Preferably, the invention also provides an application of the PD-1 specific interference plasmid in resisting melanoma.

Preferably, the invention also provides the application of the attenuated salmonella in resisting melanoma.

Compared with the prior art, the PD-1 specific interference sequence, the plasmid, the attenuated salmonella and the application in tumor resistance provided by the invention have the following beneficial effects:

we use RNAi technology to block PD-1/PD-L1 pathway from gene expression source head, and develop siRNA to overcome the problems in the background art. The salmonella serving as the carrier of the PD-1 specific interference sequence has the following advantages:

(1) salmonella has the property of accumulating in tumor tissue: compared with normal tissues, the tumor tissue can be 5000 to 10000 times higher;

(2) the intracellular invasive nature of salmonella allows it to survive within macrophages: this is closely related to its metastatic and trafficking stability in blood and body fluids and targeting to solid tumors;

(3) salmonella is a facultative anaerobe that allows it to grow in large, highly hypoxic solid tumors: but also in smaller metastases with oxygen;

therefore, the constructed attenuated salmonella carrier PD-1siRNA is used for treating tumors, so that a new strategy and a preparation are provided for treating the tumors clinically, and great market economic benefits are generated.

Drawings

FIG. 1 is a graph showing the results of Western blot detection of PD-1 expression for 24h and 48 h;

in FIG. 1, the upper and lower left panels are the results of identifying the cellular proteins of 24h, the upper and lower right panels are the results of identifying the cellular proteins of 48h, the internal reference is Tubulin, lanes 1-1 and 1-2 are the results of pH1Si-PD-1 verification, lanes 2-1 and 2-2 are the results of pH2Si-PD-1 verification, lanes 3-1 and 3-2 are the results of pH3Si-PD-1 verification, and Control is the result of the Control group;

FIG. 2 is a photograph of tumor tissues of each group of mouse animal experiments;

in FIG. 2, the left panel is PBS group, the middle panel is Scramble group, and the right panel is PD-1-siRNA group;

FIG. 3 is a western blot assay of CD8 molecule expression from groups of mouse animal experiments;

FIG. 4 is a western blot examination of the expression of PD-1 molecules for groups of mouse animal experiments;

FIG. 5 is a western blot assay of the expression of Cyclin D1 molecules from various groups of mouse animal experiments;

FIG. 6 shows CD in splenocytes of each group of mouse animal experiments⁸⁺And CD⁴⁺The cell ratio of T;

FIG. 7 is the NK cell ratio in spleen of each group of mouse animal experiment;

the left panels of FIGS. 6 and 7 are the PBS group, the middle panel is the Scramble group, and the right panel is the PD-1siRNA group.

Detailed Description

The present invention is described in detail below with reference to specific examples, but the present invention should not be construed as being limited thereto. The test methods in the following examples, which are not specified in specific conditions, are generally conducted under conventional conditions, and the steps thereof will not be described in detail since they do not relate to the invention.

The PD-1 specific interference sequence provided by the invention is sequence 1, sequence 2 or sequence 3; wherein, the sense strand of the sequence 1 is shown as SEQ ID NO.1, the antisense strand is shown as SEQ ID NO.2, the sense strand of the sequence 2 is shown as SEQ ID NO.3, the antisense strand is shown as SEQ ID NO.4, the sense strand of the sequence 3 is shown as SEQ ID NO.5, and the antisense strand is shown as SEQ ID NO. 6; the PD-1 specific interference sequence can resist tumors, particularly melanoma.

The PD-1 gene sequence is searched from NCBI, and a synthetic siRNA chain is designed before the PD-1 specific interference sequence is synthesized, wherein the design principle is as follows:

(1) selecting siRNA chains after 100 bases of PD-1 gene promoter;

(2) a siRNA strand of 5 '-AA (N19) TT-3' selected from the PD-1 gene is used as an siRNA sense strand; the siRNA sense strand sequence is the same as the selected sequence, and the siRNA antisense strand sequence is the complementary sequence of the sense strand.

(3) If 5 '-AA (N19) TT-3' is not found, then complement with 5 '-AA (N21) -3';

(4) if 5 '-AA (N21) -3' is not found, then complement with 5 '-NA (N21) -3'; (1) n in (1) to (3) may be any base, N19 represents 19 bases, and N21 represents 21 bases;

(5) in the selected siRNA strands, the GC ratio is 35-55%;

(6) if the siRNA chains meeting the requirements of (1) to (5) cannot be found out, the GC ratio is relaxed to 30 to 70 percent until the siRNA chains meeting the requirements of (1) to (4) are selected;

after the siRNA chains are selected, BamHI and HindIII sequences are respectively added at the two ends of each siRNA chain, and then PD-1 specific interference sequences are synthesized.

In the present embodiment, in order to improve efficiency, at least three siRNA strands satisfying the requirements (1) to (5), or satisfying the requirements (1) to (4) and (6) are selected as different siRNA sense strands, and corresponding antisense strands are synthesized.

Based on the same invention concept, the PD-1 specific interference plasmid carrying the PD-1 specific interference sequence is prepared by connecting the PD-1 specific interference sequence into pGCSilencoeno2.0-U6 plasmid, and the specific preparation method is as follows:

s1, synthesizing a PD-1 specific interference sequence:

the specific PD-1 interference sequence synthesized by the specific embodiment of the invention is as follows:

sequence 1:

sense strand:

5'-GATCCGGGTTTGAGCCAACCCGTCCAGTTCAAGAGACTGGACGGGTTGGCTCAAACCTTTTTTGGAAA-3', as shown in SEQ ID NO. 1;

antisense strand:

5'-AGCTTTTCCAAAAAAGGTTTGAGCCAACCCGTCCAGTCTCTTGAACTGGACGGGTTGGCTCAAACCCG-3', as shown in SEQ ID NO. 2;

sequence 2:

sense strand:

5'-GATCCGGCCGGTTTCAAGGCATGGTCATTCAAGAGATGACCATGCCTTGAAACCGGCTTTTTTGGAAA-3', as shown in SEQ ID NO. 3;

antisense strand:

5'-AGCTTTTCCAAAAAAGCCGGTTTCAAGGCATGGTCATCTCTTGAATGACCATGCCTTGAAACCGGCCG-3', as shown in SEQ ID NO. 4;

and (3) sequence:

sense strand:

5'-GATCCGGACATGAGGATGGACATTGTTTTCAAGAGAAACAATGTCCATCCTCATGTCTTTTTTGGAAA-3', as shown in SEQ ID NO. 5;

antisense strand:

5'-AGCTTTTCCAAAAAAGACATGAGGATGGACATTGTTTCTCTTGAAAACAATGTCCATCCTCATGTCCG-3', as shown in SEQ ID NO. 6;

s2, preparing PD-1 specific interference plasmid

S2 Process the preparation of PD-1 specific interfering plasmids is illustrated by sequence 1.

S21, preparing a sense strand diluent and an antisense strand diluent:

after the sense strand and the antisense strand of the siRNA (sequence 1) synthesized in S1 were dissolved, respectively, and diluted to 100 μ M, respectively, 2 oligonucleotide solutions were obtained, and then the 2 oligonucleotide solutions were diluted to a final concentration of 1 μ g/μ l, respectively, to obtain a sense strand dilution solution and an antisense strand dilution solution, respectively.

S22, annealing the sense strand diluent and the antisense strand diluent to prepare PD1-siRNA double chain:

1) reaction system: 23 mul of 1 × annealing buffer solution, 1 mul of sense strand diluent and 1 mul of antisense strand diluent;

2) reaction conditions are as follows: heating at 90 deg.C for 3min, incubating at 37 deg.C for 1h to obtain 1 PD1-siRNA double chain, and storing at-20 deg.C under the name of PD 1-siRNA-1;

s23, linearization of plasmid vector and construction of PD-1 specific interference plasmid:

after the pGCSilencer neo2.0-U6 plasmid (Shanghai Jikai GeneChem Co., Ltd.) was digested with Bam H I and HandIII, a linearized plasmid DNA fragment (i.e., a large fragment in electrophoresis) was recovered from the agarose gel, and the PD1-siRNA-1 double strand obtained in S22 was ligated to the linearized plasmid DNA fragment to obtain a PD-1 specific interference plasmid pH1 Si-PD-1.

1) The double enzyme digestion system is as follows:

incubate at 37 ℃ for 30min to obtain linearized plasmid DNA fragments, and perform agarose electrophoresis.

2) Recovering the linearized plasmid DNA fragment (the larger one in electrophoresis) by using a DNA gel recovery kit;

3) the double strand of PD1-siRNA-1 obtained from S22 is connected to the linearized plasmid DNA fragment to obtain PD-1 specific interference plasmid pH1 Si-PD-1.

This step was carried out by ligation reaction according to the instruction of DNA ligase purchased from Takara, and the reaction system was designed as follows:

the ligation was carried out overnight (ligation reaction time 12h or more) at a temperature of 4 ℃ to obtain a ligation product of PD-1 specific pH1 Si-PD-1.

S24, transforming the PD-1 specific interference plasmid into escherichia coli for storage and standby, and the specific steps are as follows:

adding 5 μ l of S23 ligation product into 100 μ l of Escherichia coli JM109 competent cells, flicking the tube bottom, mixing, and standing on ice for 30 min; adding 1ml of non-resistant LB culture medium, culturing at 220rpm for 1h, centrifuging at 8000rpm for 1min, discarding the supernatant, leaving about 100. mu.l of the resuspended cells, applying to LB plate containing 100. mu.g/ml ampicillin, and incubating at 37 ℃ for 14-16h to obtain a monoclonal for use.

S25, screening and identification of positive clones:

1) respectively picking 6 monoclonals, and inoculating the monoclonals into 10ml LB liquid culture medium containing Amp of 100 mu g/ml;

2) shaking at 150rpm at 37 deg.C, and culturing overnight;

3) the rotation speed of the shaker was adjusted to 220rpm until OD was measured₆₀₀The value is between 0.6 and 0.8, and the strains are reserved.

4) And after continuously culturing for about 3 hours, centrifuging, collecting bacteria, extracting plasmids, and performing enzyme digestion identification. The enzyme digestion system is as follows:

incubation at 37 ℃ for 30min and agarose electrophoresis for identification confirmed that the siPD-1 sequence had ligated to the vector.

S26, verification of interference function of PD-1 specific interference plasmid

Culturing competent cells of Escherichia coli JM-109 (i.e., a monoclonal identified as positive) containing the PD-1 specific interfering plasmid to obtain OD₆₀₀When the value reached 1.5, the plasmid was extracted and the concentration was determined. Paving a T lymphocyte plate, transfecting a PD-1 specific interference plasmid (PD1-siRNA-1) into the paved T cells according to the instructions of a lipo3000 kit, collecting the cells after 24 hours, extracting proteins, and detecting the expression of the PD-1 protein by using western blot, wherein the result shows that the PD-1 specific interference plasmid PD1-siRNA-1 has a certain interference effect on the T cells.

The method for preparing the PD-1 specific interference plasmid by using the sequence 2 and the sequence 3 is the same as the method for preparing the PD-1 specific interference plasmid by using the sequence 1, and the difference is that the nucleotide sequences of a sense strand and an antisense strand of the sequence 1, the sequence 2 and the sequence 3 are different. By referring to a preparation method of PD-1 specific interference plasmid pH1Si-PD-1, positive and negative chains of a sequence 2 and a sequence 3 related in S1 are respectively used as raw materials to respectively synthesize corresponding PD-1 specific interference plasmid pH2Si-PD-1 (PD 1-siRNA-2 is synthesized by the sequence 2 in S1 to prepare pH2Si-PD-1) and pH3Si-PD-1 (PD 1-siRNA-3 is synthesized by the sequence 3 in S1 to prepare pH3 Si-PD-1); the interference function of S26 proves that pH2Si-PD-1 and pH3Si-PD-1 also have certain interference effect on T cells, but the effect of pH1Si-PD-1 is best.

Based on the same inventive concept, the invention also provides an attenuated salmonella carrying the PD-1 specific interference plasmid, and the preparation method of the salmonella comprises the following steps:

a1, preparing competent cells of phoP/phoQ attenuated Salmonella (provided by the animal center of the university of Guillain) for electrotransformation; the preparation of competent cells is performed by reference to conventional molecular biology experiments, which are not described in detail herein.

A2, transforming the PD-1 specific interference plasmid into phoP/phoQ attenuated Salmonella competent cells by electrotransformation, thereby obtaining the attenuated Salmonella carrying the PD-1 specific interference plasmid.

To further validate the efficacy of the PD-1 specific interfering plasmid and the attenuated salmonella carrying it provided by the present invention, we performed the following experiments:

1. cell level interference validation

The effects of pH1Si-PD-1, pH2Si-PD-1, and pH3Si-PD-1 on T lymphocyte EL4 cells were verified respectively: the best EL4 cells were arranged at 3X 10⁵The cell protein is extracted by spreading 6-well plates per well, culturing for 14-16h, adding pH1Si-PD-1, pH2Si-PD-1 and pH3Si-PD-1, and using deionized water as a control group for 24h and 48h respectively. Expression of PD-1 was detected using Western blot. FIG. 1 is a graph showing the results of Western blot detection of PD-1 expression for 24h and 48 h; wherein, the upper and lower two graphs on the left side in FIG. 1 are the identification results of cell protein of 24h, the upper and lower two graphs on the right side are the identification results of cell protein of 48h, the internal reference is Tubulin, lanes 1-1 and 1-2 are the verification results of pH1Si-PD-1, lanes 2-1 and 2-2 are the verification results of pH2Si-PD-1, lanes 3-1 and 3-2 are the verification results of pH3Si-PD-1, and Control is the Control group result, and as can be seen from the results in FIG. 1, the specificity of PD-1 of the invention is that of PD-1The expression of PD-1 molecules on the surface of a T cell can be effectively inhibited by pH1Si-PD-1, pH2Si-PD-1 and pH3Si-PD-1, and the inhibiting effect of pH1Si-PD-1 is most obvious.

2. Mouse animal experiment

We selected the sense strand of interfering sequence 1:

5’-GATCCGGGTTTGAGCCAACCCGTCCAGTTCAAGAGACTGGACGGGTTGGCTCAAACCTTTTTTGGAAA-3’

antisense strand:

5’-AGCTTTTCCAAAAAAGGTTTGAGCCAACCCGTCCAGTCTCTTGAACTGGACGGGTTGGCTCAAACCCG-3’

animal experiments were performed.

2.1 construction of mouse melanoma model

Melanoma cells B16 were digested, resuspended, counted, and the percentage of viable cells calculated, below 98% being considered as a cell digestion failure and unusable. Preparing the qualified cells into cell suspension with serum-free culture medium at a concentration of 1 × 10⁶Each cell/ml was placed on ice and 0.2ml (2X 10) was injected subcutaneously at about 0.7cm below the right side of the back near the root of the tail⁵Individual cells). At the inoculation site, the depth should be consistent for each mouse, and injection of local skin mounds 6-8mm in diameter should be observed after tumor cell injection, if no skin mound is considered as a failure of inoculation.

2.2 Experimental procedures

7 days after inoculation of the mice with B16 cells, the mice were randomly divided into three groups: PBS group, Scramble group, PD-1-siRNA group. PBS group: injecting PBS (0.1mol/L, pH7.2) through tail vein, and injecting 1 time every 1 week for 2 times; scramble group: tail vein injection concentration of 5X 10⁵Attenuated salmonella of individual cells/ml and carrying scarmble siRNA, injected 1 time per week for 2 times; PD1-siRNA group: tail vein injection concentration of 5X 10⁵And carrying attenuated salmonella at pH1Si-PD-1, injected 1 time per week for 2 times.

(1) 14 days after the last injection, melanoma tissues of mice are separated, proteins in the tissues are respectively extracted, and the expression of PD-1 molecules in the tumor tissues is detected by a Western blot method.

(2) The number of daily survivors and tumor sizes at the end of the experiment were recorded for each group of mice and the effect of attenuated salmonella delivery pH1Si-PD-1 on the survival and tumor growth of melanoma-bearing mice was observed.

(3) Separating mouse tumor tissue, spleen and serum at different time points, and detecting CD in tumor tissue by using immunohistochemical technique with attenuated salmonella carrier pH of 1Si-PD-1⁴⁺、CD⁸⁺Expression of T cells; FACS is used for detecting CD in spleen of tumor-bearing mouse⁴⁺、CD⁸⁺And NK cells.

2.3, results of the experiment

2.3.1 attenuated Salmonella Carrier pH1Si-PD-1 is effective in inhibiting the growth of melanoma

Mice were sacrificed 14 days after the last treatment, tumor tissues of the mice were isolated, and the results of the tumor tissues of each group are shown in FIG. 2, which is a photograph of the tumor tissues of each group in the mouse animal experiment, wherein the left side of FIG. 2 is a PBS group, the middle is a Scramble group, and the right side is a PD-1-siRNA group. As can be seen in FIG. 2, the tumor tissue in the PD-1-siRNA group was minimal, and the results showed that the use of attenuated Salmonella carrier pH1Si-PD-1 was effective in melanoma growth.

2.3.2 attenuated Salmonella Carrier pH1Si-PD-1 effectively increased expression of CD8 protein in tumor tissue

The mice were sacrificed 14 days after the last treatment, tumor tissues of the mice were separated, tumor tissue proteins were extracted, and western blot detection was performed, fig. 3 is a western blot detection graph of expression of CD8 molecules by each group of mouse animal experiments, and the results show that the application of attenuated salmonella carrying pH1Si-PD-1 can effectively increase expression of CD8 molecules in melanoma tumor tissues, revealing that CD is increased⁸⁺Infiltration of T cells.

2.3.3 attenuated Salmonella Carrier pH1Si-PD-1 effectively inhibited expression of PD-1 protein in tumor tissue

The mice are sacrificed 14 days after the last treatment, tumor tissues of the mice are separated, tumor tissue proteins are extracted, and western blot detection is carried out, and fig. 4 is a western blot detection chart of PD-1 molecular expression of each group of mouse animal experiments, and the result shows that the application of attenuated salmonella carrying pH1Si-PD-1 can effectively inhibit the expression of PD-1 molecules in melanin tumor tissues, and the anti-tumor function of T lymphocytes is enhanced.

2.3.4 attenuated Salmonella Carrier pH1Si-PD-1 effectively inhibited the expression of Cyclin D1 protein in tumor tissues

The mice are sacrificed 14 days after the last treatment, tumor tissues of the mice are separated, tumor tissue proteins are extracted, and western blot detection is carried out, and fig. 5 is a western blot detection graph of the expression of Cyclin D1 molecules in each group of mouse animal experiments, and the result shows that the expression of Cyclin D1 molecules in melanin tumor tissues can be effectively inhibited by applying attenuated salmonella carrying pH1Si-PD-1, and the proliferation of tumor cells is inhibited.

2.3.5 attenuated Salmonella Carrier pH1Si-PD-1 significantly increased CD in the spleen⁸⁺And CD⁴⁺Ratio of T cells

Killing the mice 14 days after the last treatment, separating spleen tissues of the mice, separating spleen cells, and detecting CD in the spleen cells⁸⁺And CD⁴⁺Expression of T cells. FIG. 6 shows CD in splenocytes of each group of mouse animal experiments⁸⁺And CD⁴⁺T, wherein the left panel of FIG. 6 is PBS group, the middle panel is Scramble group, and the right panel is PD-1siRNA group. The results show that the use of an attenuated Salmonella carrier pH1Si-PD-1 is effective in increasing CD in the spleen⁸⁺And CD⁴⁺Ratio of T cells.

2.3.6, attenuated Salmonella Carrier pH1Si-PD-1 significantly increased the rate of NK cells in the spleen

Mice were sacrificed 14 days after the last treatment, spleen tissues of the mice were isolated, spleen cells were isolated, and NK cell expression in the spleen cells was examined. FIG. 7 shows NK cell ratios in spleens of respective groups of mouse animal experiments, the left panel of FIG. 7 is PBS group, the middle panel is Scramble group, and the right panel is PD-1siRNA group. The results show that the use of attenuated Salmonella cargo pH1Si-PD-1 was effective in increasing the rate of NK cells in the spleen.

The experiments in the series prove that the siRNA interference sequence (shown as SEQ ID NO.1-SEQ ID NO. 6) of the PD-1 which is independently designed can effectively inhibit the expression of the PD-1 of the T cell and has obvious effect of inhibiting the expression of the PD-1.

We have found thatThe siRNA interference sequence is prepared into PD-1 specific interference plasmids of pH1Si-PD-1, pH2Si-PD-1 and pH3Si-PD-1, and the recombinant plasmid of pH1Si-PD-1 is transferred into the attenuated salmonella phoP/phoQ mutant strain by an electrotransfer method. Attenuated Salmonella phoP/phoQ bacteria carrying pH1Si-PD-1 were inoculated into 100ml of LB medium (Amp, final concentration 100. mu.g/ml) and cultured overnight (12-16h) at 37 ℃. Inoculating the strain to 100ml LB medium again at a ratio of 1:100 in the next morning until OD is reached₆₀₀When it reached 0.6, it was immediately placed on ice. The concentration of Salmonella at this time is 1-1.2X 10⁹CFU/ml. The Salmonella bacteria of each group were diluted to 2X 10 by a double dilution method (with 0.1mol/L, pH7.2 PBS)⁶CFU/ml, placed on ice. Animal experiments were performed, and animals were divided into three groups: PBS group, scramble group and PD-1siRNA group. 7 days after the mice are inoculated with tumor cells, the scrambles group and the PD-1siRNA group are injected with 2 × 10 of siRNA to each mouse⁵CFU bacterial load, PBS group injected 100 u L PBS (0.1mol/L, pH7.2). The injection was boosted once more after 7 days. Survival of the mice was recorded and tumor tissue, serum, of the mice were isolated 14 days after the last injection, respectively. Detecting the tumor size of a mouse, the expression of PD-1 in tumor tissues, the expression of CD4 and CD8 molecules and the expression of proteins related to tumor cell apoptosis and metastasis; expression of the cell silverson in serum. The result shows that the attenuated salmonella carrying PD-1siRNA can obviously prolong the survival period of mice, reduce the expression of PD-1 in tumor tissues, increase the expression of CD4 and CD8 molecules, increase the expression of apoptosis-related protein caspase3, reduce the expression of transfer-related protein MMP2 and obviously improve the content of IFN gamma and TNF alpha in the serum of the mice.

Our results show that the use of attenuated salmonella delivery pH1Si-PD-1 is effective in treating breast cancer in mice, and that good therapeutic efficacy is achieved in melanoma. We will successively come out a series of products aiming at lung cancer, gastric cancer, liver cancer, melanoma and the like (all targets adapted by PD-1 antibody). As can be seen, at present, many research and development projects are carried out at home and abroad on treating tumors by aiming at the PD-1/PD-L1 pathway, but at present, no intervention and treatment are carried out at the transcription level, which is the best period for the preemptive entry of people.

It should be noted that, when the present invention relates to a numerical range, it should be understood that two endpoints of each numerical range and any value between the two endpoints can be selected, and since the steps and methods adopted are the same as those in the embodiment, in order to prevent redundancy, the present invention describes a preferred embodiment. While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Sequence listing

<120> PD-1 specific interference sequence, plasmid, attenuated salmonella and application in anti-tumor

<160>6

<170>SIPOSequenceListing 1.0

<210>1

<211>68

<212>DNA

<213> Artificial sequence

<400>1

gatccgggtt tgagccaacc cgtccagttc aagagactgg acgggttggc tcaaaccttt 60

tttggaaa 68

<210>2

<211>68

<212>DNA

<213> Artificial sequence

<400>2

agcttttcca aaaaaggttt gagccaaccc gtccagtctc ttgaactgga cgggttggct 60

caaacccg 68

<210>3

<211>68

<212>DNA

<213> Artificial sequence

<400>3

gatccggccg gtttcaaggc atggtcattc aagagatgac catgccttga aaccggcttt 60

tttggaaa 68

<210>4

<211>68

<212>DNA

<213> Artificial sequence

<400>4

agcttttcca aaaaagccgg tttcaaggca tggtcatctc ttgaatgacc atgccttgaa 60

accggccg 68

<210>5

<211>68

<212>DNA

<213> Artificial sequence

<400>5

gatccggaca tgaggatgga cattgttttc aagagaaaca atgtccatcc tcatgtcttt 60

tttggaaa 68

<210>6

<211>68

<212>DNA

<213> Artificial sequence

<400>6

agcttttcca aaaaagacat gaggatggac attgtttctc ttgaaaacaa tgtccatcct 60

catgtccg 68

Claims (7)

1. A PD-1 specific interference sequence, wherein said PD-1 specific interference sequence is seq id No.1, seq id No.2, or seq id No. 3; wherein, the sense strand of the sequence 1 is shown as SEQ ID NO.1, the antisense strand is shown as SEQ ID NO.2, the sense strand of the sequence 2 is shown as SEQ ID NO.3, the antisense strand is shown as SEQ ID NO.4, the sense strand of the sequence 3 is shown as SEQ ID NO.5, and the antisense strand is shown as SEQ ID NO. 6; the PD-1 specific interference sequence can be used for preparing anti-melanoma drugs.

2. The PD-1 specific interference sequence of claim 1, wherein the synthetic siRNA strand is designed prior to synthesis of the PD-1 specific interference sequence according to the following design rules:

(1) selecting siRNA chains after 100 bases of PD-1 gene promoter;

(2) a siRNA strand of 5 '-AA (N19) TT-3' selected from the PD-1 gene is used as an siRNA sense strand; the siRNA antisense strand sequence is a complementary sequence of a sense strand of the siRNA;

(3) if 5 '-AA (N19) TT-3' is not found, then complement with 5 '-AA (N21) -3';

(4) if 5 '-AA (N21) -3' is not found, then complement with 5 '-NA (N21) -3'; (1) n in (1) to (3) may be any base, N19 represents 19 bases, and N21 represents 21 bases;

(5) in the selected siRNA strands, the GC ratio is 35-55%;

(6) if the siRNA chains meeting the requirements of (1) to (5) cannot be found out, the GC ratio is relaxed to 30 to 70 percent until the siRNA chains meeting the requirements of (1) to (4) are selected;

after the siRNA chains are selected, BamHI and HindIII sequences are respectively added at the two ends of each siRNA chain, and then PD-1 specific interference sequences are synthesized.

3. A PD-1 specific interference plasmid carrying a PD-1 specific interference sequence according to claim 1, wherein the PD-1 specific interference plasmid is prepared by ligating the PD-1 specific interference sequence into a pGCSilencer neo2.0-U6 plasmid.

4. The PD-1 specific interference plasmid of claim 3, which is prepared by the following method:

s1, synthesizing a PD-1 specific interference sequence:

the PD-1 specific interference sequence is sequence 1, sequence 2 or sequence 3; wherein, the sense strand of the sequence 1 is shown as SEQ ID NO.1, the antisense strand is shown as SEQ ID NO.2, the sense strand of the sequence 2 is shown as SEQ ID NO.3, the antisense strand is shown as SEQ ID NO.4, the sense strand of the sequence 3 is shown as SEQ ID NO.5, and the antisense strand is shown as SEQ ID NO. 6;

s2, preparing PD-1 specific interference plasmid:

s2 step A sequence 1 is taken as an example to illustrate the preparation method of the PD-1 specific interference plasmid,

s21, preparing a sense strand diluent and an antisense strand diluent;

s22, annealing the sense strand diluent and the antisense strand diluent to prepare PD1-siRNA double chains;

s23, linearization of plasmid vector and construction of PD-1 specific interference plasmid:

after the pGCSilencer neo2.0-U6 plasmid is subjected to double enzyme digestion by Bam H I and Hand III, a linearized plasmid DNA fragment is recovered by agarose gel, and a PD1-siRNA double strand obtained from S22 is connected to the linearized plasmid DNA fragment to obtain a PD-1 specific interference plasmid pH1 Si-PD-1;

the method for preparing the PD-1 specific interference plasmid by using the sequence 2 and the sequence 3 is the same as the method for preparing the PD-1 specific interference plasmid by using the sequence 1, and the difference is that the nucleotide sequences of a sense strand and an antisense strand of the sequence 1, the sequence 2 and the sequence 3 are different.

5. An attenuated salmonella bacterium carrying the PD-1 specific interfering plasmid of claim 3.

6. The use of the PD-1 specific interfering plasmid of claim 3 in the preparation of an anti-melanoma drug.

7. Use of the attenuated salmonella of claim 5 for the preparation of an anti-melanoma medicament.

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