CN102526760A - Recombinant attenuated salmonella vaccine and pharmaceutical composition for treating solid tumors and application thereof - Google Patents
Recombinant attenuated salmonella vaccine and pharmaceutical composition for treating solid tumors and application thereof Download PDFInfo
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Abstract
The invention provides a recombinant attenuated salmonella vaccine and pharmaceutical composition for treating solid tumors and application thereof. The invention relates to application of cytokines in the prevention and the targeted treatment of the solid tumors and use of genes with interleukin2-combined hepatocyte growth factor antagonists in the preparation of drugs for the prevention and the targeted treatment of the solid tumors. The invention further relates to a gene-based pharmaceutical composition containing the interleukin2-combined hepatocyte growth factor antagonists and a method for applying the interleukin2-combined hepatocyte growth factor antagonists to a needed subject for the prevention and the targeted treatment of the solid tumors. According to the recombinant attenuated salmonella vaccine and the pharmaceutical composition for treating the solid tumors and the application thereof, the recombinant attenuated salmonella vaccine and the pharmaceutical composition can be beneficial to the prevention and the targeted treatment of various solid tumors.
Description
Technical field
The present invention relates to the oral vaccine prevention of attenuation salmonella of the eucaryon co-expression plasmid carrier of carrier's interleukin II and human hepatocyte growth factor antagonist NK4 gene simultaneously and the application of targeted therapy solid tumors, particularly relating to simultaneously, carrier's interleukin II and the dual-gene recombinant attenuated salmonella vaccine of human hepatocyte growth factor antagonist are preventing and are treating the application in the early, middle and late phase solid tumor disease.
Background technology
Malignant tumor is one of topmost disease of harm humans health and life.Up-to-date Epidemiological study result shows that China has cancer number more than 300 ten thousand now, continues the speed increase with annual 3%.Operation, radiation and chemotherapy are the main means of present treating malignant tumor.But operation only is fit to focus early stage and limitation; And " the treatment window " of radiotherapy, chemotherapy is narrower; When killing tumor cell; To the normal cell of body particularly hemopoietic and immune system also cause damage, usually cause symptoms such as anemia, hemorrhage infection, gastrointestinal reaction, alopecia, patient's quality of life is relatively poor.At present China every year because of dead about 1,300,000 people of number of cancer.See that from the angle of health economics malignant tumor is that cost is maximum, the disease that therapeutic effect is the poorest.Therefore, the treatment problem of malignant tumor is the key subjects that pharmaceutical sanitary field needs to be resolved hurrily always, and is very important for comprehensive raising China people's health level.Along with molecular biological fast development, the Biotherapeutics of tumor is approved by people gradually since the nineties.In recent years, gene therapy has become the focus of oncotherapy, mainly is to import the exogenous gene of therapeutic value is arranged, and produces the effective antitumour immunne response thereby bring out body.
(hepatocyte growth factor is the multifunctional cytokine that is produced by Interstitial cell HGF) to hepatocyte growth factor, has strong short division, tissue forms, induces migration of epithelial cells; Effects such as invasion and attack and induction of vascular generation; These all help generation and development (Zeng Q, Chen S, the You Z of tumor tissues; Et al:Hepatocyte growthfactor inhibits anoikis in head and neck squamous cell cancinoma cells byactivation of ERK and AKt signaling independ of NK-KB (J); J Biol Chem, 2002,277 (28): 25203).Date in 1997 etc. digest cracking with pancreatic elastase to recombined human HGF; Found a kind of antagonist-NK4 (Date K of comprehensive antagonism HGF bioactive functions newly; Matsumoto k, ShimuraH, et al.HGF/NK4 is a specific antagonist for pleiotrophic actions forhepatocyte growth factor (J) .FEBS Lett; 1997,420:1-6.).(be made up of N-terminal hairpin structure and 4 kringle structures of HGF α-chain by hepatocyte growth factor, antagonist HGF) as hepatocyte growth factor for NK4; Coded sequence length 1434bp; Relative molecular weight 50kDa, but it can significantly suppress growth, propagation and transfer, and angiogenesis inhibiting (Date K, the Matsumoto K of tumor cell; Shimura H; Etal.HGF/NK4 is a specific antagonist for pleiotrophic actions of hepatocyte growthfactor FEBS Lett, 1997,420:1-6; Date K, Matsumoto K, Kuba K; Et al.Inhibition oftum or growth and invasion by a four-kringle antagonist (HGF/NK4) for hepatocytegrowth factor Oncogene; 1998,17:3045-3054), experiment in vitro confirms that also NK4 can suppress the propagation of the inductive tumor cell of HGF, migrates and attack (Okasora T; Jo J I; Tabata Y.Augmented anti-tumortherapy through natural targetability of macrophages genetically engineered byNK4 plasmid DNA.Gene Ther, 2008,15 (7): 524-530).Therefore NK4 has potential application prospect in therapy of tumor.
(interleukin-2 IL-2) claims the T cell growth factor again to interleukin II, since 1976 come to light; Many scholars have carried out a large amount of research to it, find that IL-2 can strengthen cytotoxic T cell (cytotoxic tlymphocyte through improving natural killer cell and LAK cytoactive; CTL) to the lethal effect of tumor cell, thereby and activate the anti tumor immune response that the neoplasm invasiveness lymphocyte is induced body, tumor growth is stopped or the tumor body reduces [Sun Yan; Zhang Honggang; Peng Min etc. recombination leukocyte mesonium-2 treatment solid tumor and ascites III phase clinical research (J). Chinese Journal of New Drugs, 1998,7 (3): 171.]; The Biotherapeutics that with IL-2 is the basis mainly reaches inhibitions, kills tumor cell and effects a radical cure the purpose of tumor through the intrinsic antitumor mechanism of enhancing body.At present along with development of molecular biology; The IL-2 gene has become the focus of therapy of tumor; It has obtained tangible curative effect (Stewart AK, Lassam NJ, Quirt IC in breast carcinoma and melanomatous I phase clinical experiment; Et al:A denovector-mediated genedelivery of interleukin-2 in metastatic breast cancer and melanoma:results ofa phase l clintcal trial (J) .Gene Ther, 1999; 6 (3): 350.).With IL-2 is that basic biotherapy also has been widely used in treating melanoma [Arkins MB; Lotze MT; Duther JP, et al.High-dose recombinant interleukin2 therapy for patients with metastatic melanoma:analysis of 270 patients treated betweer, 1095 and 1993.JClinOncol; 1999,17 (7); 2105-2116], leukemia [Atial M; Blaise D; Marir G; Etal.Consolidation treatment of adult avutilymphoblastic leukemia:a prospective; Randomized trial comparing allogenic versus autologous bone marrowtransplantation and testing the impact of tecombnant interleukin-2 afterautologous bone mattow transplantation.BGMTGroup.Blood, 1995,86 (4): 1619-1628], renal carcinoma, gastric cancer and colorectal cancer, hepatocarcinoma etc.
The major obstacle of therapy of tumor is Antioncogene to be delivered in the tumor tissues specificity.Attenuation salmonella has direct anti-tumor activity, and has the lot of advantages of targeted therapeutic carrier.(Zheng LM such as Zheng; LuoX; Feng M, et al:Tumor amplified protein expression therapy:Salmonella as atumor-selective protein delivery vector (J.Oncol Res, 2000; 12 (3): 127-135.) use attenuation salmonella and done clinical preceding experiment; Give multiple lotus tumor (Mus, Humanmachine tumour, human colon carcinoma, pulmonary carcinoma, breast carcinoma, lung sarcoma) mice with bacterial strain, find antibacterial accumulate in tumor locus (comprising metastatic tumor) be other normal positions 200-1000 doubly.The Salmonella of attenuation of finding preclinical study can suppress the transplantation tumor growth of mice homology and different system.For this reason; This patent combines the advantage separately of tumor vaccine design both at home and abroad at present; With gene therapy, immunization therapy altogether, make up the attenuation salmonella oral vaccine of the eucaryon coexpression vector that carries IL-2 and NK4 gene simultaneously, study the effect of its targeted therapy solid tumors.
Summary of the invention
The present invention is intended to invent the attenuation salmonella of the eucaryon co-expression plasmid carrier of carrier's interleukin II and human hepatocyte growth factor antagonist NK4 gene simultaneously; Treat with oral way; To reach the purpose of prevention and targeted therapy solid tumors, particularly relate to the application of the recombinant attenuated salmonella vaccine strain of while carrier's interleukin II and human hepatocyte growth factor antagonist at the solid tumor of prevention and serious harm human healths such as early, middle and late phase colon cancer of targeted therapy and liver cancer diseases.
First purpose of the present invention is to disclose a kind of recombinant attenuated salmonella vaccine of treating solid tumor.
Second purpose of the present invention has been to disclose a kind of genomic medicine compositions of treating solid tumor.
The 3rd purpose of the present invention has been to disclose above-mentioned recombinant attenuated salmonella vaccine and the application of pharmaceutical composition in preparation treatment solid tumor drugs.
Technical scheme of the present invention is following:
A kind of recombinant attenuated salmonella vaccine of treating solid tumor, wherein, said recombinant attenuated salmonella vaccine comprises and carries interleukin II and the dual-gene eukaryon expression plasmid of hepatocyte growth factor antagonist NK4 simultaneously.
The attenuation salmonella of the treatment solid tumor described in the technique scheme is clinical vaccine attenuation salmonella bacterial strain Ty21a or other functional similarity attenuation salmonella used.
The recombinant attenuated salmonella vaccine of the treatment solid tumor described in the technique scheme, wherein, said eukaryon expression plasmid is for comprise the eukaryon expression plasmid pCMV-IL-2-IRES-NK4 of interleukin II and hepatocyte growth factor antagonist NK4 gene simultaneously.
The recombinant attenuated salmonella vaccine of the treatment solid tumor described in the technique scheme, wherein, said recombinant attenuated salmonella vaccine is reorganization attenuation salmonella TPIN.
The recombinant attenuated salmonella vaccine of the treatment solid tumor described in the technique scheme, wherein, said interleukin II is a human interleukin-12, said hepatocyte growth factor antagonist NK4 gene is a human hepatocyte growth factor antagonist NH4 gene.
The application of the recombinant attenuated salmonella vaccine of the treatment solid tumor described in the technique scheme in preparation treatment solid tumor drugs.
A kind of genomic medicine compositions of treating solid tumor, said pharmaceutical composition comprises active component and pharmaceutically acceptable carrier, wherein, said active component is the recombinant attenuated salmonella vaccine of the treatment solid tumor described in the technique scheme.
The pharmaceutical composition of the treatment solid tumor described in the technique scheme, wherein, said pharmaceutical composition is an oral capsule.
The pharmaceutical composition of the treatment solid tumor described in the technique scheme, wherein, said solid tumor is hepatocarcinoma, gastric cancer or colon cancer etc.
The application of the genomic medicine compositions of the treatment solid tumor described in the technique scheme in preparation treatment solid tumor drugs.
To play excellent curative to the targeted therapy of solid tumor behind the attenuation salmonella vaccine strain oral immunity of carrier IL-2 involved in the present invention and NK4 gene eucaryon co-expression plasmid.With respect to other tumor biotherapy agent; The topmost advantage of this vaccine strain that we invented is: one of which; Select IL-2 gene and NK4 gene associating antitumor for use: the IL-2 gene can be through improving body NK and LAK cell activity; Enhanced CT L is to the lethal effect of tumor cell, thereby and activate tumor infiltrating lymphocyte and induce the antitumor immunity of organism effect, tumor growth is stopped or tumor regression; Simultaneously, the NK4 gene can be through blocking-up human hepatocyte growth factor (hepatocyte growth factor, short vascularization effect HGF) and then anticancer increment, diffusion; IL-2 and NK4 gene act on simultaneously, and the different phase of the complicacy of tumor development is blocked, and make it can bring into play maximum GVT.They are two years old; This attenuation salmonella vaccine strains that we invented can specificly be delivered to Antioncogene in the tumor tissues; Be good to eat burst of immunity of this vaccine of vector construction simultaneously with the Salmonella; Make that administration route is convenient, safety, this vaccine also has higher safety simultaneously, can not cause damage to body.
The inventor finds that associating interleukin II regulating liver-QI cytokine antagonist NK4 has very good preventing and targeted therapy effect for solid tumor.Specifically; But preparation topical application or oral general immunity that the present invention finds simultaneously carrier's interleukin II and the recombiant plasmid of hepatocyte factor antagonist NK4 gene, recombinant attenuated Salmonella with transfection IL-2 and NK4 gene to tumor tissues or cell; Be able to produce IL-2 and NK4, thereby can play good immunoprophylaxis and targeted therapy effect for solid tumor.In addition; The present invention is through the animal model test of nude inoculation colon cancer, hepatocarcinoma tumor and other tumor cells; The attenuation salmonella of using while carrier IL-2 and NK4 gene is directly irritated the method for stomach, finds that this oral vaccine has good actual effect for targeted therapy solid tumors.In addition, the present invention finds that also the attenuation salmonella of carrier IL-2 and NK4 gene can effectively improve the immunity of nude mice, and the effect tumor tissues can effectively suppress the generation of tumor tissues and cancer beside organism's medium vessels; And the attenuation salmonella of finding carrier IL-2 and NK4 gene does not have the induced tumor risk.The present invention is based on above discovery and be accomplished.
Put it briefly, the attenuation salmonella that first aspect present invention provides carrier IL-2 and NK4 gene simultaneously is used for preventing the purposes of the medicine of targeted therapy solid tumors in preparation.Particularly, the present invention is to provide with the attenuation salmonella is that carrier carries IL-2 and the NK4 gene is used for the purposes that gene prevents the medicine of targeted therapy malignant tumor in preparation.Described malignant tumor is the particularly malignancy diseases of sickness rate such as colon cancer, hepatocarcinoma height, serious threat human health of various solid tumors.
In an embodiment of the said purposes of first aspect present invention, the attenuation salmonella of wherein said while carrier IL-2 and NK4 gene is to use with the form of eucaryon coexpression recombiant plasmid that carries IL-2 and NK4 gene or recombinant attenuated Salmonella.In other words, the attenuation salmonella of carrier IL-2 of the present invention and NK4 gene is to use with the form of the recombiant plasmid that carries IL-2 and NK4 gene simultaneously or recombinant attenuated Salmonella.
In an embodiment of the said purposes of first aspect present invention, the attenuation salmonella of wherein said carrier's interleukin II and hepatocyte growth factor antagonist gene is human interleukin-12 and human hepatocyte growth factor antagonist.Interleukin II of the present invention can also use Mus or other mammiferous interleukin II certainly; The hepatocyte growth factor antagonist also can use Mus or other mammiferous hepatocyte growth factor antagonist.But the present invention is end user's interleukin II and human hepatocyte growth factor antagonist preferably, comprises all classification of human interleukin-12 and human hepatocyte growth factor antagonist, for example subclass, inferior group, subgroup, subtribe etc.
In an embodiment of the said purposes of first aspect present invention, wherein said recombiant plasmid is that pCMV-IL-2-IRES-NK4 or other carry the eukaryon expression plasmid of IL-2 and NK4 gene.
In an embodiment of the said purposes of first aspect present invention, wherein said attenuation salmonella is clinical vaccine strains Ty21a or other functional similarity attenuation salmonella used.
In an embodiment of the said purposes of first aspect present invention, wherein said solid tumor comprises the particularly malignancy disease of sickness rate such as colon cancer, hepatocarcinoma height, serious threat human health of various entity tumors.
Second aspect present invention provides a kind of pharmaceutical composition, and it comprises interleukin II and the combination formulations of hepatocyte growth factor antagonist and the pharmaceutically acceptable carrier of choosing wantonly of treating effective dose.According to the present invention, described pharmaceutical composition can be used for prevention and/or targeted therapy entity tumor.Described entity tumor is the malignant tumor of sickness rate such as colon cancer, hepatocarcinoma height, serious threat human health particularly.
In an embodiment of the said pharmaceutical composition of second aspect present invention, wherein said interleukin II and hepatocyte growth factor antagonist are to be present in this pharmaceutical composition with recombiant plasmid that carries interleukin II and hepatocyte growth factor antagonist gene or recombinant attenuated Salmonella form.
In an embodiment of the said pharmaceutical composition of second aspect present invention, wherein said interleukin II and hepatocyte growth factor antagonist are human interleukin-12 and human hepatocyte growth factor antagonist.
In an embodiment of the said pharmaceutical composition of second aspect present invention, wherein said recombiant plasmid is that pCMV-IL-2-IRES-NK4 or other carry the eukaryon expression plasmid of IL-2 and NK4 gene simultaneously.
In an embodiment of the said pharmaceutical composition of second aspect present invention, wherein said recombinant attenuated Salmonella is clinical vaccine strains Ty21a or other functional similarity attenuation salmonella used.
According to the pharmaceutical composition of second aspect present invention, it can be used for preventing and/or the targeted therapy entity tumor high malignant tumor of sickness rate such as colon cancer, hepatocarcinoma particularly.
In an embodiment of the said pharmaceutical composition of second aspect present invention, wherein said solid tumor is serious threat human healths such as phase morning, noon and afternoon colon cancer, hepatocarcinoma, malignant tumor that sickness rate is high particularly.
According to each said pharmaceutical composition of second aspect present invention, it is an oral capsule etc.
Third aspect present invention provide a kind of in the experimenter of needs the prevention and/or the method for targeted therapy entity tumor, this method comprises to the interleukin II of said experimenter's administering therapeutic effective dose and the combination formulations of hepatocyte growth factor antagonist.
In an embodiment of the said method of third aspect present invention, wherein said interleukin II and hepatocyte growth factor antagonist are to use with the form of recombiant plasmid that carries interleukin II and hepatocyte growth factor antagonist gene simultaneously or recombinant attenuated Salmonella.
In an embodiment of the said method of third aspect present invention, wherein said interleukin II and hepatocyte growth factor antagonist are human interleukin-12 and human hepatocyte growth factor antagonist.Interleukin II of the present invention and hepatocyte growth factor antagonist can also use Mus or other mammiferous interleukin II and hepatocyte growth factor antagonist certainly.But the present invention is end user's interleukin II and hepatocyte growth factor antagonist preferably.
In an embodiment of the said method of third aspect present invention, wherein said recombiant plasmid is that pCMV-IL-2-IRES-NK4 or other carry the eukaryon expression plasmid of IL-2 and NK4 gene simultaneously.
In an embodiment of the said method of third aspect present invention, wherein said recombinant attenuated Salmonella is clinical vaccine strains Ty21a or other functional similarity attenuation salmonella used.
In an embodiment of the said method of third aspect present invention, wherein said solid tumor comprises the particularly malignant tumor of sickness rate such as colon cancer, hepatocarcinoma height, serious threat human health of various entity tumors.
Hereinafter, the present invention also provides the medical application of interleukin II and hepatocyte growth factor antagonist gene combination formulations.
The combination formulations that fourth aspect present invention provides interleukin II and hepatocyte growth factor antagonist is used for preventing and/or the purposes of targeted therapy solid tumors medicine in preparation.Particularly, the combination formulations that the present invention is to provide interleukin II and hepatocyte growth factor antagonist is used for the purposes of gene prevention and/or targeted therapy solid tumors medicine in preparation.Described solid tumor is the malignant tumor of sickness rate such as colon cancer, hepatocarcinoma height, serious threat human health particularly.
Be described in further detail in the face of the present invention down.
Implication in general sense as well known to those skilled in the art, that the term " prevention " that the present invention uses and " targeted therapy " have them.
The term " interleukin II " that the present invention uses is meant for example people's interleukin II of mammal.The preferred interleukin II of the present invention is a human interleukin-12.It will be apparent to those skilled in the art that " human interleukin-12 " has implication well known in the art.
The term " hepatocyte growth factor antagonist " that the present invention uses is meant for example people's hepatocyte growth factor antagonist of mammal.The preferred hepatocyte growth factor of the present invention is a human hepatocyte growth factor.It will be apparent to those skilled in the art that " human hepatocyte growth factor " has implication well known in the art.
The term " TPIN " that the present invention uses is meant the attenuation salmonella vaccine strain that carries IL-2 gene and NK4 gene eucaryon co-expression plasmid carrier simultaneously.TPIN of the present invention possesses tumor-targeting, and the efficient of transfection interleukin II and NK4 gene is higher than plasmid, and only at IL-2 of merocrine secretion and NK4, does not have to get into blood flow and cause or the dangerous and misgivings of induced tumor development therefore have practical value.In addition; The attenuation salmonella TPIN that it is considered herein that associating IL-2 and NK4 gene has the immunologic function of adjusting simultaneously and suppresses the effect of tumor tissues angiogenic growth and have the therapeutic effect to solid tumor; For this reason; The present invention carries out the animal experiment of nude inoculation tumor cell, and uses TPIN and directly irritate the method observation of stomach and identify curative effect, achieves tangible results.
In the present invention, described interleukin II and hepatocyte growth factor antagonist are to use with the form of the recombiant plasmid that carries interleukin II and hepatocyte growth factor antagonist gene simultaneously or recombinant attenuated Salmonella.The plasmid that inserts is the eukaryon expression plasmid of ammonia benzyl resistance, for example the pCMV-IL-2-IRES-NK4 that makes up voluntarily of the inventor, the recombinant attenuated Salmonella bacterial strain TPIN of the attenuation salmonella Ty21a of the eucaryon co-expression plasmid that carries IL-2 and NK4 gene that makes up of the inventor for example.
The invention provides a kind of novel method that is used for targeted therapy solid tumors, this method comprises the recombiant plasmid or the recombinant attenuated Salmonella of carrying interleukin II and hepatocyte growth factor antagonist gene simultaneously, treats this disease thereby give the cancer patient.Solid tumor comprises a variety of, as: the malignant entity tumor of serious threat human healths such as hepatocarcinoma, colon cancer etc.According to spirit of the present invention, the method for treating above-mentioned entity tumor also is provided, this method comprises the recombiant plasmid or the recombinant attenuated Salmonella of carrying interleukin II and hepatocyte growth factor antagonist gene, treats this disease thereby give the cancer patient.
Except interleukin II and hepatocyte growth factor antagonist, also can contain medicinal adjuvant commonly used in the pharmacy well known by persons skilled in the art as required, for example excipient, stabilizing agent, antiseptic, carrier etc. in the pharmaceutical composition of the present invention.
The present invention has following beneficial effect:
Use Therapeutic Method provided by the invention and have more science than other treatment means, targeting property, the curative effect of safety and oncotherapy are clearer and more definite.Under the situation of present needleless better clinical treatment means still to solid tumor; The invention provides a kind of new effective Therapeutic Method; Therapeutic Method of the present invention has more science than treatment and prevention of tumour means in the past, and targeting property is better, safe, curative effect is clearer and more definite.
Description of drawings:
1, Fig. 1 is the pcr amplification of IL-2 gene;
2, Fig. 2 is the pcr amplification of NK4 gene;
3, Fig. 3 identifies figure for the pCMV-IL-2 enzyme action;
4, Fig. 4 is the enzyme action evaluation figure of pCMV-IL-2-IRES-NK4 plasmid;
5, Fig. 5 is the PCR screening of reorganization attenuation salmonella TPIN;
6, Fig. 6 is TPIN group and a Ty21a group tumor body diameter comparison diagram behind the tumor cell inoculation 2W;
7, Fig. 7 detects TPIN group and the scattergram of Ty21a group eukaryotic expression promoter CMV in tissue for PCR;
8, Fig. 8 detects TPIN group and the distribution of Ty21a group eukaryotic expression promoter CMV in tissue for PCR;
9, Fig. 9 respectively organizes IL-2 gene expression dose in the nude mice tissue gene for PCR detects;
10, Figure 10 respectively organizes NK4 gene expression dose in the nude mice tissue gene for PCR detects.
The specific embodiment:
For making technical scheme of the present invention be convenient to understand, the present invention is further described below in conjunction with the specific embodiment.
For making technical scheme of the present invention be convenient to understand, the present invention is further described below in conjunction with the specific embodiment.
Further specify the present invention through specific embodiment below, still, be to be understood that into, these embodiment are only used for the usefulness of explanation more in detail particularly, are used for limiting in any form the present invention and should not be construed as.These embodiment the preparation of the recombiant plasmid that carries interleukin II and hepatocyte growth factor antagonist gene simultaneously or recombinant attenuated Salmonella is described and to the therapeutical effect after the nude mice implanted tumor cells with further elaboration the present invention.
The present invention carries out generality and/or concrete description to the material and the test method that are used in the test.Though for realizing that employed many materials of the object of the invention and operational approach are well known in the art, the present invention still does to describe in detail as far as possible at this.It will be apparent to those skilled in the art that hereinafter, if do not specify that material therefor of the present invention and operational approach are well known in the art.
(1) carries the recombiant plasmid of interleukin II and hepatocyte growth factor antagonist NK4 gene
The structure of pCMV-IL-2-IRES-NK4 and recombinant attenuated Salmonella TPIN and preparation
(A) clone of IL-2 cDNA
Press bibliographical information (Eizenberg; O; Et al.R Interleukin-2 transcripts in human and rodentbrains:possible expression by astrocytes.J.Neurochem.1995,64 (5): human interleukin-12 1928-1936) (interleukin-2, IL-2) cDNA sequential design primer; In forward primer, introduce the XhoI restriction enzyme site, introduce MLu I restriction enzyme site in the reverse primer.The oligonucleotide sequence of forward primer is 5-gacctcgagatgtacaggatgcaactc-3; The oligonucleotide sequence of reverse primer is 5-cgaacgcgttcacagtgttgagatgatgc-3; Use conventional Protocols in Molecular Biology; From human placenta cDNA library (available from Clontech company, the U.S.), pass through human cloning interleukin II (IL-2) gene in the polymerase chain reaction,PCR (PCR).The PCR parameter is: 94 ℃ of preparatory degeneration 5min, and 94 ℃ of 30s, 48 ℃ of 30s, 72 ℃ of 1min, period is 30,72 ℃ and extends 10min.The PCR product (as shown in Figure 1, M:Maker2000 wherein; The 1:PCR product) measures human IL-2 cDNA sequence (worker's biotechnology service company is given birth in Shanghai) with the two deoxidation cessation method of Sanger.The human IL-2 cDNA sequence of human IL-2 cDNA sequence that records and bibliographical information compared to analyze show; The human IL-2 cDNA sequence of our clone's human IL-2 cDNA sequence and bibliographical information is in full accord, and IL-2 cDNA (444bp) sequence is in the sequence table shown in the sequence 1.
(B) clone of NK4 cDNA
Go up hepatocyte growth factor (hepatocyte growth factor, HGF)) the antagonist NK4 cDNA sequential design primer of announcing according to PUBMED, in forward primer, introduce Sal I restriction enzyme site, introduce Not I restriction enzyme site in the reverse primer.The oligonucleotide sequence of forward primer is 5-CTG
GTCGACATG TGG GTG ACC AAA CTC-3, the oligonucleotide sequence of reverse primer is 5-GCA
GCGGCCGCTCAG ACT ATT GTA GGT GTG GT-3; Amplification obtains NK4 cDNA sequence from the pcKH plasmid; The pcKH plasmid had before made up for this laboratory; Its building process is following: use conventional Protocols in Molecular Biology, (forward primer is 5 '-ccatcgatgttaacatgtgggtgaccaaactc-3 ' through polymerase chain reaction,PCR (PCR) from human placenta cDNA library (available from Clontech company, the U.S.); Downstream primer is 5 '-tgggatccgcggccgcctatgactgtggtacctt-3 ') separate and to obtain people HGF coding region cDNA (2184bp); Analysis-by-synthesis is also carried out in order-checking, and the people HGF full length cDNA sequence that the people HGF full length cDNA sequence that records and Gene Bank have been announced compares to analyze and shows that the people HGF full length cDNA sequence that people HGFcDNA sequence that we clone and Gene Bank have announced is in full accord; Then its sub-clone is changed voluntarily MCS (the BamH I of the carrier for expression of eukaryon pcK of structure to us; Apa I site), obtain the recombinant vector pcKH of carrier's liver cell growth factor gene, human hepatocyte growth factor gene receives the CMV promoter regulation.PcK is (available from Invitrogen company with commercial carrier pcDNA3; The U.S.) ammonia Bian resistant gene is changed to kalamycin resistance gene and changes the structure gained; The pcDNA3 carrier with SspI (available from Promega) and AvrII (available from Promega) double digestion, is cut the ammonia benzyl because of (about 0.6kb), and pEGFP-N1 (available from Invitrogen) uses the AvrII enzyme action; Reclaim big fragment (about 4.8kb) and the small fragment (dna fragmentation of 1.1kb respectively; This fragment contains complete kanamycin gene) connect with T4 dna ligase (available from BioLabs), obtain the pcDNA3 carrier of kalamycin resistance, called after pcK; Use BamHI (available from Promega) and ApaI (available from Promega) double digestion pcK and pSK-HGF respectively; Reclaim big fragment and the small fragment (dna fragmentation of 2.2kb respectively; This fragment contains complete people HGF gene) connect with the T4 dna ligase; Obtain the carrier for expression of eukaryon of carrier's liver cell growth factor gene, contain kalamycin resistance gene, called after pcKH.
Be that amplification template passes through polymerase chain reaction (PCR) method amplification and obtains the NK4 gene with pcKH, the PCR parameter is: 94 ℃ of preparatory degeneration 5min, and 94 ℃ of 30s, 54 ℃ of 30s, 72 ℃ of 2min, period is 30,72 ℃ and extends 10min.PCR product (as shown in Figure 2, M:Maker2000 wherein, 1:PCR product) is measured people NK4 cDNA sequence (worker's biotechnology service company is given birth in Shanghai) with the two deoxidation cessation method of Sanger.The people NK4 cDNA sequence of people NK4 cDNA sequence that records and bibliographical information compared to analyze show; The people NK4 cDNA sequence of our clone's people NK4 cDNA sequence and bibliographical information is in full accord, and NK4 cDNA sequence (1434bp) is in the sequence table shown in the sequence 2.
(C) preparation of the carrier for expression of eukaryon pCMV-IL-2 of carrier's interleukin II gene
With pIRES-SEQ carrier (available from Invitrogen company) with Xho I (available from TaKaRa) and MLu I (available from TaKaRa) double digestion; Use Xho I (available from TaKaRa) and MLu I (available from TaKaRa) the double digestion interleukin II gene of purification simultaneously; Enzyme action reclaimed linearizing pIRES plasmid fragment (about 6.1kb) and interleukin II genetic fragment (490bp) respectively with 1% gel electrophoresis (gel reclaims test kit, available from Promega) after 3 hours; PIRES-SEQ that reclaims and IL-2 mix with mole ratio at 7: 1; Add the T4 ligase and connect Buffer; 4 ℃ connect 12h, obtain the carrier for expression of eukaryon of carrier's liver cell growth factor gene, contain ampicillin resistance gene; Called after pCMV-IL-2 (as shown in Figure 3, M:250bp DNA Ladder wherein; 1:pIRES-SEQ Xhol single endonuclease digestion; 2:pCMV-IL-2 Xhol single endonuclease digestion; 3:pCMV-IL-2 plasmid Xhol, Mlu1 double digestion).
Use ordinary skill in the art method; The pCMV-IL-2 plasmid is transformed conventional competence antibacterial (for example bacillus coli DH 5 alpha), large scale fermentation antibacterial amplification plasmid then, centrifugal recovery thalline; Adopt alkaline denaturation to extract recombiant plasmid; (adopt the big extraction reagent kit of no endotoxin plasmid, TIANGEN), agarose gel electrophoresis and ultraviolet spectrophotometry are confirmed purity and the content of DNA to the column chromatography large scale plasmid purification.Obtain required plasmid and be used for following experiment.
(D) the eucaryon coexpression vector of carrier's interleukin II gene and hepatocyte growth factor antagonist NK4 gene
The preparation of pCMV-IL-2-IRES-NK4
With the pCMV-IL-2 carrier that makes up in the step (C) with Sal I (available from TaKaRa) and Not I (available from TaKaRa) double digestion; Use Sal I (available from TaKaRa) and Not I (available from TaKaRa) the double digestion hepatocyte growth factor antagonist NK4 gene of purification simultaneously; Enzyme action reclaimed linearizing pCMV-IL-2 plasmid fragment (about 6.6kb) and hepatocyte growth factor antagonist NK4 genetic fragment (1471bp) respectively with 1% gel electrophoresis (gel reclaims test kit, available from Promega) after 3 hours; The pCMV-IL-2 that reclaims and NK4 are with mole ratio 5～10: 1 mixing; Add the T4 ligase and connect Buffer; 4 ℃ connect 12h, obtain the carrier for expression of eukaryon of carrier's liver cell growth factor gene, contain ampicillin resistance gene; Called after pCMV-IL-2-IRES-NK4 (identify like Fig. 4, wherein M:250bp DNA Ladder by enzyme action; 1:pCMV-IL-2 Sal1 single endonuclease digestion; 2:pCMV-IL-2-IRES-NK4 Sal1 single endonuclease digestion; 3:pCMV-IL-2-IRES-NK4 plasmid Sal1, Not1 double digestion).
Use ordinary skill in the art method; Plasmid pCMV-IL-2-IRES-NK4 is transformed conventional competence antibacterial (for example bacillus coli DH 5 alpha), large scale fermentation antibacterial amplification plasmid then, centrifugal recovery thalline; Adopt alkaline denaturation to extract recombiant plasmid; (adopt the big extraction reagent kit of no endotoxin plasmid, TIANGEN), agarose gel electrophoresis and ultraviolet spectrophotometry are confirmed purity and the content of DNA to the column chromatography large scale plasmid purification.Obtain required plasmid and be used for following experiment.
(E) preparation of recombinant attenuated Salmonella TPIN
I, electroporation transform
The frozen bacterium liquid 50 μ L of attenuation salmonella Ty21a are inoculated in 5ml do not contain antibiotic LB culture fluid, concussion is cultured to logarithmic growth mid-term (bacterium liquid A value about 0.4), under 4 ℃ of conditions, and 4000r.min
-1Centrifugal collection thalline, with the sterile deionized water washed cell of pre-cooling 2 times, the antibacterial after the washing is suspended from the sterile deionized water of 1ml ice pre-cooling.With electroporation (Multiporator 4308 Eppendorf electroporations) pCMV-IL-2-IRES-NK4 plasmid 0.2 μ g is transformed the pretreated attenuation salmonella of 200 μ L.Electroporation conditions: voltage 2.5kV, electric capacity 25 μ F, resistance 200 Ω, discharge time 0.5s.Add SOC culture medium 1ml, 45min is cultivated in 37 ℃ of soft concussions, coats to contain ampicillin (100mg.L
-1) solid LB culture medium plate, 2 bacterium colonies of each picking are identified behind 37 ℃ of cultivation 16h.
The screening of ii, positive engineering bacteria
From 2 single bacterium colonies of culture plate picking, be inoculated in 3mL respectively and contain in the LB culture fluid of kanamycin, 37 ℃ jolt cultivation.Amicillin resistance is passed through in the screening that changes the attenuation salmonella bacterial strain of pCMV-IL-2-IRES-NK4 over to, (the PCR screening is as shown in Figure 5, wherein M:250bp DNA Ladder for PCR; 1:TPIN PCR identifies the IL-2 gene; 2:TPINPCR evaluation NK4 gene) XhoL, Mlu1 double digestion behind (utilizing bacterium liquid to be template, amplification eukaryotic expression promoter CMV and genes of interest IL-2, NK4) and the extraction plasmid, Sal1, Not1 double digestion are identified.The primer of amplification eukaryotic expression promoter CMV is: the upper reaches 5 '-ccc agt aca tga cct tat ggg-3 ', and downstream 5 '-gga gac ttg gaa atcccc gt-3 ', the PCR parameter is: 94 ℃ of 30sec; 55 ℃ of 30sec; 72 ℃ of 1min, period is 30,72 ℃ and extends 5min.The primer of amplification IL-2 is: the upper reaches 5 '-gacctcgagatgtacaggatgcaactc-3 '; Downstream 5 '-cgaacgcgttcacagtgttgagatgatgc-3 ', the PCR parameter is: 94 ℃ of preparatory degeneration 5min, 94 ℃ of 30sec; 48 ℃ of 30sec; 72 ℃ of 60sec, period is 30,72 ℃ and extends 10min eventually.The primer of amplification NK4 is: the upper reaches 5 '-CTG
GTCGACATG TGG GTG ACC AAA CTC-3 ', downstream 5 '-GCA
GCGGCCGCTCAGACT ATT GTA GGT GTG GT-3 ', the PCR parameter is: 94 ℃ of preparatory degeneration 5min, 94 ℃ of 30sec, 56 ℃ of 30sec, 72 ℃ of 2min, period is 30,72 ℃ and extends 10min.
The preparation of iii, engineering bacteria TPIN
The attenuation salmonella strain (TPIN) of the eucaryon coexpression vector that carries IL-2 and NK4 gene is inoculated in the LB culture medium that contains ampicillin (50 μ g/mL) 35～37 ℃ of 8%CO
2Cultivate 24h in the incubator as generation strain; With amplification culture in generation bacterial classification inoculation to the Kolle flask that contains complete medium as secondary strain; And under aseptic condition, get generation strain and secondary strain appearance smear; Microscopically is visible behind the Gram is gram negative bacilli, and long-chain is many, and bacterium shape is consistent.Secondary Kolle flask strain is scraped in 100mL sterile saline seed bottle, is 1.7 * 10 than turbid result
10Individual/mL, the centrifugal 20min of 3000r/min behind the 30L ferment tank 12h collects thalline 73g, adds the abundant mixing of 70mL skim milk, places the stainless steel disc lyophilization, is prepared into the about 23g of sterile dry.
Below through the pharmacodynamic experiment of TPIN the beneficial effect that the present invention has is described:
Test Example 1:IL-2 and NK4 reorganization eucaryon co-expression plasmid pCMV-IL-2-IRES-NK4 and recombinant attenuated Salmonella
The effect observation of bacterium TPIN in the hepatoma-targeting treatment
(1) TPIN is to nude mice prevention of hcc effect observation
(1) laboratory animal and model:
30 of the nude mouses that Gansu Province college of traditional Chinese medicine Experimental Animal Center provides, male, body weight 60～80 grams.Experiment is bought the last week, freely drinks water and ingests.HEPG
2In containing the RPMI1640 culture medium of 10% calf serum, cultivate.Condition of culture is 37 ℃ of 5%CO
2Treat 6 weeks of mice oral immunity, blank group (Blank), TPIN group, Ty21a group are respectively got 10 of nude mouses, and every in right subcutaneous abdomen inoculation 5 * 10
5Individual HEPG
2Cell.
(2) application of TPIN:
The model nude mice is divided into three groups at random: be respectively blank group (Blank), TPIN group, Ty21a group.TPIN, Ty21a are inoculated in 2mL respectively contain in the LB culture fluid of 2uLAmp and antibiotic-free, shaken overnight is got 50uL adding 50mL next day and is contained in the corresponding antibiotic LB culture fluid, collects thalline behind the 2h, and PBS uses 10% NaHCO after cleaning
3Suspend, the adjustment bacterial population is 1 * 10
9/ mL.Each experimental mice is raised the corresponding antibacterial 0.1mL of clothes with stomach tube, Blank group clothes equivalent 10% NaHCO
3Solution.2 weeks 1 time, totally three times.
(3) lymphocyte subgroup analysis:
Oral immunity is done peripheral blood CD3+CD4+, the analysis of CD3+CD8+T lymphocyte subgroup after 6 weeks.Compare with Ty21a group, blank group, CD4+, CD4+/CD8+ ratio have marked difference (P＜0.01) in the TPIN immune group peripheral blood.Also have obviously with Ty21a group, blank group comparison TPIN immune group CD4+, CD4+/CD8+ ratio and to increase (P＜0.05).Ty21a immune group and blank group comparison CD4+/CD8+ ratio increase (P＜0.05).The result sees table 1.
Behind table 1 oral immunity to the influence
of mice peripheral blood CD4 ' T, CD8 ' T, CD4 '/CD8 ' ratio
Annotate:
*: TPIN compares P＜0.01 with blank control group,
*: Ty21a compares P＜0.05 with blank control group.
(4) IgM, IgG in the whole blood
1
, the IgA level
IgM, IgG in the TPIN immune group peripheral blood
1, IgA level and blank group significantly raise (P＜0.01); Compare IgM, IgG with the Ty21a group
1Level significantly raises (P＜0.05), and the IgA level also raises, but not statistically significant (P＞0.05) (table 2).
Behind table 2 oral immunity to the influence
of mice peripheral blood Immunoglobulin IgM, IgG1, IgA
Annotate:
*: TPIN compares P＜0.01 with blank control group,
*: Ty21a compares P＜0.05 with blank control group.
(4) to the influence of nude mice spleen lymphocyte proliferation
Each organizes splenocyte under LPS, PHA and different immunogen stimulate, and the SI value all has variation in various degree.TPIN immune group and blank group, Ty21a organize relatively, splenocyte proliferation activity be significantly increased (P＜0.05) under Ty21a, TPIN, LPS, the effect of PHA stimulus object; Ty21a group and blank group relatively, Ty21a,, splenocyte proliferation activity be significantly increased (P＜0.05) (table 3) under the LPS, PHA, the effect of TPIN stimulus object.
Behind table 3 oral immunity to the influence
of mice T, B cell proliferation
Annotate:
*: TPI compares P＜0.01 with blank control group,
*: Ty21a compares P＜0.05 with blank control group.
(5) experiment conclusion:
We discover that CD4+ in the TPIN immune group nude mice peripheral blood, CD4+/CD8+ ratio are than Ty21a group, blank group be significantly increased (P＜0.01).Compare IgM, IgG with the blank group
1, the IgA level has significantly and increases (P＜0.05); Compare IgM, IgG with the Ty21a group
1Level significantly increases (P＜0.05), and the IgA level reduces, but not statistically significant.We adopt mtt assay to study it to the mouse boosting cell Immune Effects, and we find TPIN immune group and blank group relatively, and the splenocyte proliferation activity is significantly increased under Typ21a, TPIN, LPS, PHA condition.Compare with the Typ21a group, the splenocyte proliferation activity is significantly increased under TPIN, LPS, PHA condition.This explanation TPIN can improve immune level in the nude mouse effectively, activates intravital antitumor mechanism, thereby helps suppressing the propagation and the diffusion of tumor cell.
(2) TPIN is to the inhibition and the therapeutical effect of liver cancer tissue growth
(1) laboratory animal and model:
30 of the nude mouses that Gansu Province college of traditional Chinese medicine Experimental Animal Center provides, male, body weight 60～80 grams.Experiment is bought the last week, freely drinks water and ingests.HEPG
2In containing the RPMI1640 culture medium of 10% calf serum, cultivate.Condition of culture is 37 ℃ of 5%CO
2Treat 6 weeks of mice oral immunity, blank group (Blank), TPIN group, Ty21a group are respectively got 10 of nude mouses, and every in right subcutaneous abdomen inoculation 5 * 10
5Individual HEPG
2Cell.
(2) application of TPIN:
The model nude mice is divided into four groups at random: be respectively blank group (Blank), TPIN group, Ty21a group.TPIN, Ty21a are inoculated in 2mL respectively contain in the LB culture fluid of 2uLAmp, shaken overnight is got 50uL adding 50mL next day and is contained in the corresponding antibiotic LB culture fluid, collects thalline behind the 2h, and PBS uses 10% NaHCO after cleaning
3Suspend, the adjustment bacterial population is 1 * 10
9/ mL.Each experimental mice is raised the corresponding antibacterial 0.1mL of clothes with stomach tube, Blank group clothes equivalent 10% NaHCG
3Solution.2 weeks 1 time, totally three times.
(3) variation of observation tumor growth:
Behind the tumor cell inoculation 2W, mice takes off neck puts to death, and isolates the tumor body, the vernier caliper measurement diameter.Ty21a group and blank group average tumor diameter are (5.1 ± 0.6) mm, (5.5 ± 0.7) mm, no significant difference property between two groups (P＞0.05); TPIN group average tumor diameter is (2.8 ± 0.4) mm, is significantly less than preceding two groups (P＜0.01) (result is as shown in Figure 6).
(5) experiment conclusion:
The mice of oral immunity is behind the inoculated tumour cell, and the size of its tumor cell is significantly less than blank control group, and this explanation IL-2 can activate corresponding immunization route in vivo, brings into play its antitumor action thereby NK4 possibly can suppress the tumor tissues vessel growth.Therefore, can prove that oral TPIN can improve mouse immune power effectively, thereby suppress the growth that the tumor tissues vessel growth suppresses tumor tissues.
(3) TPIN is to the targeting property assessment of tumor tissues
(1) laboratory animal and model:
30 of the nude mouses that Gansu Province college of traditional Chinese medicine Experimental Animal Center provides, male, body weight 60～80 grams.Experiment is bought the last week, freely drinks water and ingests.HEPG
2In containing the RPMI1640 culture medium of 10% calf serum, cultivate.Condition of culture is 37 ℃ of 5%CO
2Treat 6 weeks of mice oral immunity, blank group (Blank), TPIN group, Ty21a group are respectively got 10 of nude mouses, and every in right subcutaneous abdomen inoculation 5 * 10
5Individual HEPG
2Cell.
(2) application of TPIN:
The model nude mice is divided into three groups at random: be respectively blank group (Blank), TPIN group, Ty21a group.TPIN, Ty21a are inoculated in 2mL respectively contain in the LB culture fluid of 2uLAmp, shaken overnight is got 50uL adding 50mL next day and is contained in the corresponding antibiotic LB culture fluid, collects thalline behind the 2h, and PBS uses 10% NaHCO after cleaning
3Suspend, the adjustment bacterial population is 1 * 10
9/ mL.Each experimental mice is raised the corresponding antibacterial 0.1mL of clothes with stomach tube, Blank group clothes equivalent 10% NaHCO
3Solution.2 weeks 1 time, totally three times.
(3) PCR testing goal expression of gene is assessed the targeting property of TPIN to tumor tissues:
Carrier for expression of eukaryon pCMV-IL-2-IRES-NK4 is promoter with CMV, detects to organize with PCR to have or not the CMV fragment to exist in the genome.The result shows that TPIN organizes the fragment that all can amplify an about 140bp size in the liver of mice, spleen, stomach, small intestinal, kidney, the tumor tissues; The CMV clip size of estimating during with design of primers conforms to, and (as shown in Figure 7, wherein 1:TPIN group tumor tissues genome is a template; 2:TPIN group spleen tissue gene group is a template; 3:TPIN group hepatic tissue genome is a template; 4:TPIN group gastric tissue genome is a template; 5:TPIN group nephridial tissue genome is a template; 6:TPIN group small intestine genome is a template; 7:2000 DNA Marker; 8:Ty21a group tumor tissues genome is a template; 9:Ty21a group spleen tissue gene group is a template; 10:Ty21a group hepatic tissue genome is a template; 11:Ty21a group gastric tissue genome is a template; 12:Ty21a group nephridial tissue genome is a template; 13:Ty21a group small intestine genome is a template).And do not see the fragment of 140bp size in the liver of nude mice of control group, spleen, stomach, small intestinal, kidney, tumor tissues.
(5) experiment conclusion:
PCR check explanation attenuation salmonella can carry the exogenous gene group really and get in the nude mouse; And further in the histiocyte genome, integrate and express; And can have certain targeting property in the tumor locus enrichment, be suitable as the carrier of oral antioncogene medicine.
Test Example 2: the effect observation of recombinant attenuated Salmonella TPIN in the nude mice target treatment of colon cancer
(1) TPIN's observes nude mice colon cancer preventive effect
(1) laboratory animal and model:
30 of the nude mouses that Gansu Province college of traditional Chinese medicine Experimental Animal Center provides, male, body weight 60～80 grams.Experiment is bought the last week, freely drinks water and ingests.The LoVo colon cancer cell is cultivated in containing the RPMI1640 culture medium of 10% calf serum.Condition of culture is 37 ℃ of 5%CO
2Treat 6 weeks of mice oral immunity, blank group (Blank), TPIN group, Ty21a group are respectively got 10 of nude mouses, and every in right subcutaneous abdomen inoculation 5 * 10
5Individual LoVo cell.
(2) application of TPIN:
The model nude mice is divided into three groups at random: be respectively blank group (Blank), TPIN group, Ty21a group.TPIN, Ty21a are inoculated in 2mL respectively contain in the LB culture fluid of 2uLAmp, shaken overnight is got 50uL adding 50mL next day and is contained in the corresponding antibiotic LB culture fluid, collects thalline behind the 2h, and PBS uses 10% NaHCO after cleaning
3Suspend, the adjustment bacterial population is 1 * 10
9/ mL.Each experimental mice is raised the corresponding antibacterial 0.1mL of clothes with stomach tube, Blank group clothes equivalent 10% NaHCO
3Solution.2 weeks 1 time, totally three times.
(3) lymphocyte subgroup analysis:
Oral immunity is done peripheral blood CD3+CD4+, the analysis of CD3+CD8+T lymphocyte subgroup after 6 weeks.Compare with Ty21a group, blank group, CD4+, CD4+/CD8+ ratio have marked difference (P＜0.01) in the TPIN immune group peripheral blood.Also have obviously with Ty21a group, blank group comparison TPIN immune group CD4+, CD4+/CD8+ ratio and to increase (P＜0.05).Ty21a immune group and blank group comparison CD4+/CD8+ ratio increase (P＜0.05).The result sees table 4.
Behind table 4 oral immunity to the influence
of mice peripheral blood CD4 ' T, CD8 ' T, CD4 '/CD8 ' ratio
Annotate:
*: SPIN compares P＜0.01 with blank control group,
*: Ty21a compares P＜0.05 with blank control group.
(4) IgM, IgG in the whole blood
1
, the IgA level
IgM, IgG in the TPIN immune group peripheral blood
1, IgA level and blank group significantly raise (P＜0.01); Compare IgM, IgG with the Ty21a group
1Level significantly raises (P＜0.05), and the IgA level also raises, but not statistically significant (P＞0.05) (table 5).
Behind table 5 oral immunity to the influence
of mice peripheral blood Immunoglobulin IgM, IgG1, IgA
Annotate:
*: TPIN compares P＜0.01 with blank control group,
*: Ty21a compares P＜0.05 with blank control group.
(5) to the influence of nude mice spleen lymphocyte proliferation
Each organizes splenocyte under LPS, PHA and different immunogen stimulate, and the SI value all has variation in various degree.TPIN immune group and blank group, Ty21a organize relatively, splenocyte proliferation activity be significantly increased (P＜0.05) under Ty21a, TPIN, LPS, the effect of PHA stimulus object; Ty21a group and blank group relatively, Ty21a,, splenocyte proliferation activity be significantly increased (P＜0.05) (table 6) under the LPS, PHA, the effect of TPIN stimulus object.
Behind table 6 oral immunity to the influence
of mice T, B cell proliferation
Annotate:
*: TPIN compares P＜0.01 with blank control group,
*: Ty21a compares P＜0.05 with blank control group.
(6) experiment conclusion:
We discover that CD4+ in the TPIN immune group nude mice peripheral blood, CD4+/CD8+ ratio are than Ty21a group, blank group be significantly increased (P＜0.01).Compare IgM, IgG with the blank group
1, the IgA level has significantly and increases (P＜0.05); Compare IgM, IgG with the Ty21a group
1Level significantly increases (P＜0.05), and the IgA level reduces, but not statistically significant.We adopt mtt assay to study it to the mouse boosting cell Immune Effects, and we find TPIN immune group and blank group relatively, and the splenocyte proliferation activity is significantly increased under Typ21a, TPIN, LPS, PHA condition.Compare with the Typ21a group, the splenocyte proliferation activity is significantly increased under TPIN, LPS, PHA condition.This explanation TPIN can improve immune level in the nude mouse effectively, activates intravital antitumor mechanism, thereby helps suppressing the propagation and the diffusion of tumor cell.
(2) TPIN is to the inhibition and the therapeutical effect of colon cancer growth
(1) laboratory animal and model:
30 of the nude mouses that Gansu Province college of traditional Chinese medicine Experimental Animal Center provides, male, body weight 60～80 grams.Experiment is bought the last week, freely drinks water and ingests.The LoVo cell is cultivated in containing the RPMI1640 culture medium of 10% calf serum.Condition of culture is 37 ℃ of 5%CO
2Treat 6 weeks of mice oral immunity, blank group (Blank), TPIN group, Ty21a group are respectively got 10 of nude mouses, and every in right subcutaneous abdomen inoculation 5 * 10
5Individual LoVo cell.
(2) application of TPIN:
The model nude mice is divided into four groups at random: be respectively blank group (Blank), TPIN group, Ty21a group.TPIN, Ty21a are inoculated in 2mL respectively contain in the LB culture fluid of 2uLAmp and antibiotic-free, shaken overnight is got 50uL adding 50mL next day and is contained in the corresponding antibiotic LB culture fluid, collects thalline behind the 2h, and PBS uses 10% NaHCO after cleaning
3Suspend, the adjustment bacterial population is 1 * 10
9/ mL.Each experimental mice is raised the corresponding antibacterial 0.1mL of clothes with stomach tube, Blank group clothes equivalent 10% NaHCO
3Solution.2 weeks 1 time, totally three times.
(3) variation of observation tumor growth:
Behind the tumor cell inoculation 2W, mice takes off neck puts to death, and isolates the tumor body, the vernier caliper measurement diameter.Ty21a group and blank group average tumor diameter are (5.0 ± 0.2) mm, (5.3 ± 0.5) mm, no significant difference property between two groups (P＞0.05); TPIN group average tumor diameter is (3.0 ± 0.4) mm, is significantly less than preceding two groups (P＜0.01).
(5) experiment conclusion:
Oral TPIN can improve mouse immune power effectively; Thereby suppress the generation of tumor tissues vessel growth prophylaxis of tumours; Experimental result shows that the TPIN group obviously diminishes than the matched group tumor tissues; Significant difference proves that IL-2 can activate corresponding immunization route in vivo, brings into play its antitumor action thereby NK4 possibly can suppress the tumor tissues vessel growth.Therefore, can prove that oral TPIN can improve mouse immune power effectively, thereby suppress the growth that the tumor tissues vessel growth suppresses tumor tissues.
(3) TPIN is to the targeting property assessment of tumor tissues
(1) laboratory animal and model:
40 of the nude mouses that Gansu Province college of traditional Chinese medicine Experimental Animal Center provides, male, body weight 60～80 grams.Experiment is bought the last week, freely drinks water and ingests.HEPG
2In containing the RPMI1640 culture medium of 10% calf serum, cultivate.Condition of culture is 37 ℃ of 5%CO
2Treat 6 weeks of mice oral immunity, blank group (Blank), TPIN group, Ty21a group are respectively got 5 of nude mouses, and every in right subcutaneous abdomen inoculation 5 * 10
5Individual LoVo cell.
(2) application of TPIN:
The model nude mice is divided into three groups at random: be respectively blank group (Blank), TPIN group, Ty21a group.TPIN, Ty21a are inoculated in 2mL respectively contain in the LB culture fluid of 2uLAmp, shaken overnight is got 50uL adding 50mL next day and is contained in the corresponding antibiotic LB culture fluid, collects thalline behind the 2h, and PBS uses 10% NaHCO after cleaning
3Suspend, the adjustment bacterial population is 1 * 10
9/ mL.Each experimental mice is raised the corresponding antibacterial 0.1mL of clothes with stomach tube, Blank group clothes equivalent 10% NaHCO
3Solution.2 weeks 1 time, totally three times.
(3) PCR testing goal expression of gene is assessed the targeting property of TPIN to tumor tissues:
Carrier for expression of eukaryon pCMV-IL-2-IRES-NK4 is promoter with CMV, detects to organize with PCR to have or not the CMV fragment to exist in the genome.The result shows that TPIN organizes the fragment that all can amplify an about 140bp size in the liver of mice, spleen, stomach, small intestinal, kidney, the tumor tissues; The CMV clip size of estimating during with design of primers conforms to, and (as shown in Figure 8, wherein 1:TPIN group tumor tissues genome is a template; 2:TPIN group spleen tissue gene group is a template; 3:TPIN group hepatic tissue genome is a template; 4:TPIN group gastric tissue genome is a template; 5:TPIN group nephridial tissue genome is a template; 6:TPIN group small intestine genome is a template; 7:2000 DNA Marker; 8:Ty21a group tumor tissues genome is a template; 9:Ty21a group spleen tissue gene group is a template; 10:Ty21a group hepatic tissue genome is a template; 11:Ty21a group gastric tissue genome is a template; 12:Ty21a group nephridial tissue genome is a template; 13:Ty21a group small intestine genome is a template).And do not see the fragment of 140bp size in the liver of nude mice of control group, spleen, stomach, small intestinal, kidney, tumor tissues.
(4) IL-2 of PCR testing goal gene and NK4 gene are in the intravital expression of nude mice:
Attenuation salmonella carries IL2 and NK4 genetic immunization nude mice, detects IL-2 and the NK4 expression of gene situation in the nude mice tissue gene group respectively organized with PCR.The result shows in the liver, spleen, stomach, small intestinal, kidney, tumor tissues of TPIN group mice all can amplify fragment big or small about an about 500bp and 1500bp, and the IL-2 that estimates during with design of primers (as shown in Figure 9,1:marker2000 wherein; 2:TPIN group IL-2 expresses; 3:Ty21a group IL-2 expresses; 4: matched group IL-2 expresses; 5:marker2000; 6:TPIN group β-actin expresses; 7:Ty21a group β-actin expresses; 8: matched group β-actin expresses) and NK4 (wherein 1:marker2000 shown in figure 10; 2:TPIN group NK4 gene expression (on), β-actin expresses (descending); 3:Ty21a group group NK4 gene expression (on), β-actin expresses (descending); 4: matched group group NK4 gene expression (on), β-actin expresses (descending); 5:marker2000) the genetic fragment size conforms to, and IL-2 and NK4 expression of gene level obviously raise than matched group.
(5) experiment conclusion:
PCR check explanation attenuation salmonella can carry the exogenous gene group really and get in the nude mouse; And further in the histiocyte genome, integrate and express; And can have certain targeting property in the tumor locus enrichment, be suitable as the carrier of oral antioncogene medicine.
Experimental result shows that the recombiant plasmid recombinant salmonella can effectively treat malignant entity tumors such as hepatocarcinoma, colon cancer.According to above presentation of results, the attenuation salmonella TPIN oral vaccine strain that contains interleukin II and hepatocyte growth factor antagonist NK4 gene when the present invention relates to has the effect of prevention and targeted therapy solid tumors really.
The above; Being merely preferred embodiment of the present invention, is not that the present invention is done any formal and substantial restriction, allly is familiar with the professional and technical personnel; In not breaking away from technical scheme scope of the present invention; The technology contents that is disclosed more than capable of using, and a little change of making, modify the equivalent variations with differentiation, be equivalent embodiment of the present invention; Simultaneously, all foundations essence technology of the present invention all still belongs in the scope of technical scheme of the present invention change, modification and the differentiation of any equivalent variations that above embodiment did.
Claims (10)
1. recombinant attenuated salmonella vaccine of treating solid tumor is characterized in that: said recombinant attenuated salmonella vaccine comprises and carries interleukin II and the dual-gene eukaryon expression plasmid of hepatocyte growth factor antagonist NK4 simultaneously.
2. the recombinant attenuated salmonella vaccine of treatment solid tumor according to claim 1 is characterized in that: said attenuation salmonella is attenuation salmonella Ty21a or other functional similarity attenuation salmonellas.
3. the recombinant attenuated salmonella vaccine of treatment solid tumor according to claim 1 is characterized in that: said eukaryon expression plasmid is for comprise the eukaryon expression plasmid pCMV-IL-2-IRES-NK4 of interleukin II and hepatocyte growth factor antagonist NK4 gene simultaneously.
4. the recombinant attenuated salmonella vaccine of treatment solid tumor according to claim 1 is characterized in that: said recombinant attenuated salmonella vaccine is reorganization attenuation salmonella TPIN.
5. according to the recombinant attenuated salmonella vaccine of the described anti-treatment solid tumor of claim 1～4, it is characterized in that: interleukin II is a human interleukin-12, and hepatocyte growth factor antagonist NK4 gene is a human hepatocyte growth factor antagonist NH4 gene.
6. the application of the recombinant attenuated salmonella vaccine of the described treatment solid tumor of arbitrary claim in preparation treatment solid tumor drugs in the claim 1～5.
7. pharmaceutical composition of treating solid tumor, said pharmaceutical composition comprises active component and pharmaceutically acceptable carrier, it is characterized in that: said active component is the described recombinant attenuated salmonella vaccine of arbitrary claim in the claim 1～5.
8. the pharmaceutical composition of treatment solid tumor according to claim 7 is characterized in that: said pharmaceutical composition is an oral capsule.
9. according to the pharmaceutical composition of claim 7 or 8 described treatment solid tumors, it is characterized in that: said solid tumor is hepatocarcinoma, gastric cancer or colon cancer etc.
10. the application of the pharmaceutical composition of the described treatment solid tumor of arbitrary claim in preparation treatment solid tumor drugs in the claim 7～9.
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| CN113766927A (en) * | 2019-02-27 | 2021-12-07 | 总医院公司 | Treatment of benign nervous system tumors using attenuated salmonella typhimurium |
| CN114073777A (en) * | 2020-08-18 | 2022-02-22 | 中国科学院深圳先进技术研究院 | Bacterial targeting vector carrying cytokine or polynucleotide thereof and application thereof in tumor treatment |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113766927A (en) * | 2019-02-27 | 2021-12-07 | 总医院公司 | Treatment of benign nervous system tumors using attenuated salmonella typhimurium |
| CN114073777A (en) * | 2020-08-18 | 2022-02-22 | 中国科学院深圳先进技术研究院 | Bacterial targeting vector carrying cytokine or polynucleotide thereof and application thereof in tumor treatment |
| WO2022036618A1 (en) * | 2020-08-18 | 2022-02-24 | 中国科学院深圳先进技术研究院 | Bacterial targeting vector carrying cytokine or polynucleotide thereof and use thereof in tumor treatment |
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