CN1221795A

CN1221795A - Human interleukin 6 core transcription factor expression plasmid series and its application in treating tumour

Info

Publication number: CN1221795A
Application number: CN 98122022
Authority: CN
Inventors: 刘定干
Original assignee: Shanghai Institute of Biochemistry
Current assignee: Shanghai Institutes for Biological Sciences SIBS of CAS
Priority date: 1998-11-20
Filing date: 1998-11-20
Publication date: 1999-07-07
Anticipated expiration: 2018-11-20
Also published as: CN1163613C

Abstract

The present invention relates to a plasmid DNA-human interleukin 6 nuclear transcription factor expressing plasmid series, which can suppress the growth of cancer in human body, so it can be used to prevent and cure various cancers.

Description

Human interleukin 6 nuclear transcription factor expression plasmid series and its application in tumor treatment

The present invention relates to the field of gene engineering and gene therapy, and is especially one kind of human interleukin-6 nuclear transcription factor (NF-IL6) expressing plasmid series and its application in treating tumor.

The existing tumor treatment medicines have large toxicity and side effects (such as chemotherapeutic medicines) and low effects (such as Chinese herbal medicines and preparations thereof), so that the tumor can not be satisfactorily controlled and the life of a patient can not be saved.

The present inventors found in research work that the artificial expression of exogenous human interleukin 6 nuclear transcription factor in some malignant cells causes apoptosis of these malignant cells, and the work is published in [ Zhu Min-sheng and Liu Ding-gan et al, DNA and Cell Biology,1997,16(2):127-135 ]. International also published the results of studies in which NF-IL6 is essential for macrophages to exert their bactericidal and malignant Cell function [ Tanaka, T.et al, Cell,1995,80(2):353-361 ]. The results of these studies indicate that the NF-IL6 gene may be used to treat malignancies.

In order to allow the NF-IL6 gene to be expressed in malignant cells and related effector cells, it is necessary to link it to a plasmid vector. Plasmid vectors are DNA, a tool currently commonly used in molecular biology and genetic engineering research; they have all the elements that enable the expression of the artificially inserted gene (i.e.transcription into the messenger ribonucleic acid [ mRNA ], followed by synthesis of a physiologically acting protein according to the genetic information provided by the mRNA) (see, for example, Sambrook et al, Molecular cloning: A Laboratory Manual.2nd ed., Cold Spring Harbor Laboratory, 1989). Therefore, it is of great significance and practical value to design and construct expression plasmid series containing NF-IL6 gene and research the application of the expression plasmid series in tumor treatment.

The present invention aims at providing NF-IL6 expression plasmid series, which can inhibit the growth of in vivo tumor and can be used for treating tumor.

The NF-IL6 expression plasmid series (also called as '14-6 RS') provided by the invention is designed and constructed according to the research results of the inventor and the world on tumor phenotype and gene expression, and has the following basic structure:

the basic structure 1 is shown in figure 1, and is a circular double-stranded DNA molecule formed by sequentially connecting a promoter, an NF-IL6 coding region, a DNA sequence 1, a polyA signal and a DNA sequence 2, wherein:

NF-IL6 coding region = coding region for human interleukin 6 nuclear transcription gene;

promoter = the promoter of any eukaryotic gene, which is an element that promotes expression of the gene immediately downstream thereof, such as the SV40 virus early promoter; the SV40 virus late promoter; a cytomegalovirus promoter; a promoter of a metallothionein gene; a promoter for the myosin gene; an adenovirus promoter; a vaccinia virus promoter; a herpes virus promoter; an influenza virus promoter; a promoter of actin gene; a promoter for a collagen gene; a papillomavirus promoter; a polyoma viral promoter; a promoter of an alpha-fetoprotein gene; a promoter for a carcinoembryonic antigen gene; mouse Mammary Tumor Virus (MMTV) promoter, etc.

DNA sequence 1= DNA sequence of any eukaryotic plasmid vector between the multiple cloning site and the polyA signal, typically containing splicing signals for mRNA (messenger ribonucleic acid);

polyA signal = polyA signal of any eukaryotic plasmid vector, which is the start site of polyA (poly adenine nucleotide) tail of mRNA;

DNA sequence 2= DNA sequence of any eukaryotic plasmid vector between the polyA signal and the promoter, which typically contains the replication initiation site of the plasmid vector DNA, and may also contain antibiotic resistance genes, including genes that confer resistance to certain antibiotics to bacteria (e.g., ampicillin resistance gene amp, tetracycline resistance gene tet, kanamycin resistance gene kan, etc.), or genes that confer resistance to certain antibiotics to eukaryotic cells (e.g., antibiotic G418 resistance gene neo, etc.).

Any of the eukaryotic plasmid vectors described above, for example, the following plasmids: SPORT1, pSPORT1, pMSEx-1, pMSEx-2, pMSEx-3, pcDNA3, pSFV1, pCDV-1, pSVK3, pBPV, pMSG, etc.

The basic structure 2 is shown in FIG. 2, and is a linear double-stranded DNA molecule formed by sequentially connecting a DNA sequence 2 (one part), a promoter, an NF-IL6 coding region, a DNA sequence 1, a polyA signal and a DNA sequence 2 (the other part), wherein:

DNA sequence 2= the DNA sequence of any eukaryotic plasmid vector downstream of the polyA signal and/or upstream of the promoter, typically contains the replication initiation site of the plasmid vector DNA, and may also contain antibiotic resistance genes, including genes that confer resistance to certain antibiotics to the bacterium (e.g., ampicillin resistance gene amp, tetracycline resistance gene tet, kanamycin resistance gene kan, etc.), or genes that confer resistance to certain antibiotics to eukaryotic cells (e.g., antibiotic G418 resistance gene neo, etc.).

The NF-IL6 expression plasmid series is constructed by using a genetic engineering method. Cutting a plasmid vector containing a promoter, a polyA site, a plasmid replication origin, an antibiotic resistance gene and other regulatory sequences into a linear shape by using an appropriate restriction enzyme; the NF-IL6 coding region was also excised from the plasmid cloned with the NF-IL6 gene using restriction enzymes, purified and appropriately treated; then connecting the two into a ring by using DNA ligase, transforming bacteria, amplifying, extracting plasmid and purifying to obtain the DNA-based DNA-. The final product is pure plasmid DNA, free of any proteins, polysaccharides (including pyrogens).

If the purified plasmid is cut in the region of the DNA sequence 2 by using proper restriction enzyme, a linear plasmid is obtained.

The preparation methods will be described in detail in the following examples, taking pSN, pCN, pVN, pTN and pGN as examples. The 14-6RS of the other structure can be prepared by a general genetic engineering method with reference to this example.

The 14-6RS takes pSN as an example and is proved to be capable of effectively inhibiting the growth of tumor and even leading the tumor to disappear and recover without obvious side effect through treatment experiments on human tumor loaded by experimental animals. For example, a tumor formed by subcutaneously inoculating human liver cancer cells to a nude mouse, 1-10 micrograms of 14-6RS is injected into each tumor once, the tumor can be observed to gradually shrink and tend to disappear after 3 days, and the tumor of a control is increased; after the nude mice are inoculated with human gastric cancer cells in the abdominal cavity, 10 micrograms of 14-6RS is injected into the abdominal cavity of each mouse once, after half a month, a contrast person is bled in the abdominal cavity, the tumor is large and necrotic through dissection, the contrast person injected with 14-6RS shows 1/3 has no lesion through dissection, and 2/3 tumor is obviously smaller than the contrast; tumors formed by subcutaneous inoculation of human lung cancer cells in nude mice were injected with 1-10 micrograms of "14-6 RS" per tumor, and significantly slower tumor proliferation was observed compared to controls.

Thus, the NF-IL6 expression plasmid series of the present invention is a plasmid DNA having a function of shrinking and/or eliminating a tumor in vivo. The expression plasmid series of the present invention is constructed by using plasmid vectors containing different promoters, so that the expression plasmid series can treat various malignant tumors in different tissues, for example, the expression plasmid containing the alpha-fetoprotein promoter is particularly suitable for treating liver cancer. When it was injected into or near the tumor site of nude mice bearing various human malignant tumors, the tumor was significantly reduced or even disappeared as compared with the control. The effective dose is very low, and in tumor-bearing nude mice, the tumor proliferation can be inhibited by injecting several micrograms of each tumor. In observations to date, animals injected with the "14-6 RS" did not experience significant toxic reactions and drug side effects. Therefore, the 14-6RS can be developed into a high-efficiency and safe gene medicine for treating and preventing various tumors.

Description of the drawings:

FIG. 1 is the basic structure 1 (circular) of the NF-IL6 expression plasmid series.

FIG. 2 is the basic structure 2 (linear) of the NF-IL6 expression plasmid series.

FIG. 3 is a flowchart of a method for constructing plasmid pSN.

FIG. 4 is a flow chart of a method for constructing plasmid pCN.

FIG. 5 is a flow chart of a method for constructing plasmid pVN.

FIG. 6 is a flowchart of a method for constructing plasmid pTN.

FIG. 7 is a flow chart of the method of construction of plasmid pGN.

FIG. 8 is a graph showing the results of a control group and a treatment curve of plasmid pSN on human hepatoma nude mouse tumor.

FIG. 9 shows the experimental results of the treatment of tumor implantation of plasmid pSN in nude mice with gastric cancer in humans (1), in the frame of mice: the right side is the effective nude mouse, and the left side is the dying nude mouse.

FIG. 10 shows the experiment (2) for treating tumor implantation of plasmid pSN in nude mice with human gastric cancer, the dissection of nude mice: the right side is the effective treatment nude mouse, and the left side is the dead nude mouse.

FIG. 11 is a graph of the treatment result curve of plasmid pSN on human lung cancer nude mouse hybridoma and a control group.

The invention is further described by the following examples, without limiting the scope of the invention.

Example 1 construction of plasmid pSN A preparation of plasmid vector and NF-IL6 Gene fragment

1. Plasmid pMSEx-l (present at doctor Schuemann, Germany, containing SV40 virus early promoter) 0.5. mu.g (0.5. mu.l) was added to 10 Xrestriction enzyme StuI buffer solution 2. mu.l, sterilized water 17. mu.l and restriction enzyme StuI 1. mu.l, mixed, and incubated at 37 ℃ for 1 hour for enzyme reaction. The reaction solution was extracted once with phenol/chloroform and once with chloroform, and the plasmid was precipitated with ethanol and dissolved in 10. mu.l of 10mmol/L Tris.HClpH8.0-1 mmol/L EDTA.

2. Mu.l of the above dissolved pMSEx-19. mu.l, 1. mu.l of 10 Xcalf intestinal alkaline phosphatase buffer solution and 0.5. mu.l of calf intestinal alkaline phosphatase were taken, mixed and incubated at 37 ℃ for 30 minutes to carry out an enzymatic reaction. The reaction solution was added with 1. mu.l of 50m mol/L EDTA, heated at 75 ℃ for 10 minutes, and then extracted once with phenol/chloroform and once with chloroform, and the plasmid was precipitated with ethanol and dissolved in 10. mu.l of 10m mol/L Tris.HCl pH8.0-1m mol/L EDTA to obtain solution A.

3. 0.5. mu.g (0.5. mu.l) of plasmid pBlue610 (present by Japan, Hospital, Male Bosch), 2. mu.l of 10 Xrestriction enzyme SalI buffer, 17. mu.l of sterile water and 1. mu.l of restriction enzyme SalI were taken, mixed, and incubated at 37 ℃ for 1 hour to carry out an enzymatic reaction. Mu.l of a solution containing 10mmol/L each of dATP, dCTP, dTTP and dGTP and 1. mu. L T4 DNA polymerase were added to the reaction solution, and incubation was continued for 20 minutes. The reaction solution was separated by 0.7% low melting point agarose gel electrophoresis, the 1.0kb band was excised, the plasmid DNA was extracted from the gel, 1 time by phenol/chloroform extraction, once by chloroform extraction, the plasmid was precipitated with ethanol, and dissolved in 10. mu.l of 10mmol/L Tris.HCl pH8.0-1mmol/L EDTA as solution B, the blunt-ended NF-IL6 coding region. b. Ligation of the blunt NF-IL6 coding region with the plasmid vector pMSEx-1, bacterial transformation and characterization

Mu.l of solution A, 10. mu.l of solution B, 2. mu.l of 10XT4 DNA ligase buffer, 2. mu.l of 10m mol/L ATP and 2. mu.l of T4 DNA ligase were mixed and incubated overnight at 4 ℃. The next day, 2. mu.l of the reaction solution was taken to transform E.coli DH 5. alpha. and spread on LB solid medium containing 100. mu.g/ml ampicillin, cultured overnight, colonies were picked up randomly, 1ml of LB liquid medium was used to amplify the bacteria, plasmids were extracted, and digested with restriction enzyme EcoRI, respectively, the reaction solution was separated by 0.7% normal agarose gel electrophoresis, and the plasmid with 1.3kb insert was selected, namely plasmid pSN. Can be stored and amplified for later use.

The above process is shown in fig. 3.

Example 2 construction of plasmid pCN A preparation of plasmid vector and NF-IL6 Gene fragment

1. Plasmid pcDNA3 (from Invitrogen, containing SV40 virus early promoter) was used in an amount of 0.5. mu.g (0.5. mu.l), 2. mu.l of 10 Xrestriction enzyme EcoRI buffer, 16. mu.l of sterile water, 1. mu.l of restriction enzyme EcoRI and 1. mu.l of restriction enzyme XhoI, and mixed and incubated at 37 ℃ for 1 hour to carry out the enzymatic reaction. The reaction solution was extracted once with phenol/chloroform and once with chloroform, and the plasmid was precipitated with ethanol and dissolved in 10. mu.l of 10mmol/L Tris.HCl pH8.0-1mmol/L EDTA to obtain solution A.

2. Plasmid pBlue 6100.5. mu.g (0.5. mu.l), 10 Xrestriction enzyme EcoRI buffer 2. mu.l, sterilized water 17. mu.l, restriction enzyme EcoRI 1. mu.l and restriction enzyme XhoI 1. mu.l were taken, mixed, and incubated at 37 ℃ for 1 hour for enzyme reaction. The reaction mixture was separated by 0.7% low melting point agarose gel electrophoresis, the 1.0kb band was excised, and plasmid DNA was extracted from the gel and dissolved in 10. mu.l of 10mmol/L Tris.HCl pH8.0-1mmol/LEDTA as solution B, i.e., NF-IL6 coding region. Ligation and bacterial transformation of the NF-IL6 coding region with the plasmid vector pcDNA3

Mu.l of solution A, 10. mu.l of solution B, 2. mu.l of 10XT4 DNA ligase buffer, 2. mu.l of 10m mol/L ATP and 1. mu.l of T4 DNA ligase were mixed and incubated at 16 ℃ overnight for enzymatic reaction. The next day, 1. mu.l of the reaction mixture was used to transform E.coli DH 5. alpha. and spread on LB solid medium containing 100. mu.g/ml ampicillin, overnight, colonies were randomly picked up, 1ml of LB liquid medium was used to amplify the bacteria, plasmids were extracted, each plasmid was double digested with restriction enzymes EcoRI and XhoI, the reaction mixture was separated by 0.7% normal agarose gel electrophoresis, and a plasmid containing an insert of about 1.0kb was selected (without further identification), which was plasmid pCN. Can be stored and amplified for later use.

The above process is shown in fig. 4.

Example 3 construction of plasmid pVN a preparation of plasmid vector and NF-IL6 Gene fragment

1. Plasmid pSV.SPORT1 (purchased from GibcoBRL, containing SV40 virus early promoter) was used in an amount of 0.5. mu.g (0.5. mu.l), 10 Xrestriction enzyme SalI buffer (2. mu.l), sterilized water (17. mu.l) and restriction enzyme SalI (1. mu.l), and the mixture was incubated at 37 ℃ for 1 hour to carry out enzymatic reaction. The reaction solution was extracted once with phenol/chloroform and once with chloroform, and the plasmid was precipitated with ethanol and dissolved in 10. mu.l of 10mmol/L Tris.HCl pH8.0-1mmol/L EDTA.

2. Mu.l of the above-dissolved pSV.SPORT19, 1. mu.l of 10 Xcalf intestinal alkaline phosphatase buffer and 0.5. mu.l of calf intestinal alkaline phosphatase were mixed and incubated at 37 ℃ for 30 minutes to carry out an enzymatic reaction. The reaction solution was added with 1. mu.l of 50m mol/L EDTA, heated at 75 ℃ for 10 minutes, and then extracted once with phenol/chloroform and once with chloroform, and the plasmid was precipitated with ethanol and dissolved in 10. mu.l of 10m mol/L Tris.HCl pH8.0-1m mol/L EDTA to obtain solution A.

3. Plasmid pBlue 6100.5. mu.g (0.5. mu.l), 10 Xrestriction enzyme SalI buffer 2. mu.l, sterilized water 17. mu.l and restriction enzyme SalI 1. mu.l were taken, mixed, and incubated at 37 ℃ for 1 hour to carry out an enzymatic reaction. The reaction mixture was separated by 0.7% low melting point agarose gel electrophoresis, the 1.0kb band was excised, and plasmid DNA was extracted from the gel and dissolved in 10. mu.l of 10mmol/L Tris.HCl pH8.0-1mmol/L EDTA as solution B, i.e., the NF-IL6 coding region. Ligation of NF-IL6 coding region and plasmid vector pSV.SPORT1, bacterial transformation and characterization

Mu.l of the solution A, 10. mu.l of the solution B, 2. mu.l of 10XT4 DNA ligase buffer, 2. mu.l of 10mmol/LATP and 1. mu.l of T4 DNA ligase were mixed and incubated at 16 ℃ overnight for enzymatic reaction. The next day, 1. mu.l of the reaction solution was taken to transform E.coli DH 5. alpha. and spread on LB solid medium containing 100. mu.g/ml ampicillin, cultured overnight, colonies were picked up randomly, 1ml of LB liquid medium was used to amplify the bacteria, plasmids were extracted, and each was subjected to enzymatic hydrolysis with the restriction enzyme SalI, the reaction solution was separated by 0.7% normal agarose gel electrophoresis, and plasmids showing about 1.0kb of insert fragment were selected and further purified for identification. c. Identification of Forward inserted recombinant plasmids

And c, taking 9 mu l of the recombinant plasmid obtained in the step b, adding 1 mu l of 10XEcoRI enzyme buffer solution and 4 units of EcoRI enzyme, mixing, preserving the temperature at 37 ℃ for 2 hours to carry out enzyme reaction, separating the reaction solution by using 0.7% common agarose gel electrophoresis, and selecting the recombinant plasmid without a fragment of about 1.0kb, namely the plasmid pVN. Can be stored and amplified for later use.

The above process is illustrated in fig. 5.

EXAMPLE 4 construction of plasmid pTN a preparation of plasmid vector and NF-IL6 Gene fragment

1. A plasmid pSMT (professor Shenzhou Shen of institute of animals, national academy of sciences) containing a metallothionein gene promoter in an amount of 0.5. mu.g (0.5. mu.l), 2. mu.l of 10 Xrestriction enzyme buffer A (Boehringer-Mannheim), 17. mu.l of sterilized water and 1. mu.l of restriction enzyme SmaI were mixed, incubated at 25 ℃ for 2 hours for enzymatic reaction, then 1. mu.l of restriction enzyme KpnI was added thereto, and incubation at 37 ℃ was continued for 2 hours. The reaction solution was extracted 1 time with phenol/chloroform, once with chloroform, and the plasmid was precipitated with ethanol, dissolved in 10. mu.l of 10m mol/L Tris.HCl pH8.0-1m mol/L EDTA, as solution A.

2. Mu.g (0.5. mu.l) of plasmid pBlue 6100.5, 10 Xrestriction enzyme buffer A2. mu.l, 17. mu.l of sterilized water and 1. mu.l of restriction enzyme SmaI were mixed, incubated at 25 ℃ for 2 hours to carry out the enzyme reaction, then 1. mu.l of restriction enzyme KpnI was added, and incubation at 37 ℃ was continued for 2 hours. The reaction mixture was separated by 0.7% low melting point agarose gel electrophoresis, the 1.0kb band was excised, and plasmid DNA was extracted from the gel and dissolved in 10. mu.l of 10mmol/L Tris.HCl pH8.0-1mmol/L EDTA as solution B, i.e., the NF-IL6 coding region. Ligation and bacterial transformation of the NF-IL6 coding region and the plasmid vector pSMT

Mu.l of solution A, 10. mu.l of solution B, 2. mu.l of 10XT4 DNA ligase buffer, 2. mu.l of 10m mol/L ATP and 1. mu.l of T4 DNA ligase were mixed and incubated at 4 ℃ overnight for enzymatic reaction. The next day, 1. mu.l of the reaction mixture was used to transform E.coli DH 5. alpha. and spread on LB solid medium containing 100. mu.g/ml ampicillin, overnight, colonies were randomly picked up, 1ml of LB liquid medium was used to amplify the bacteria, plasmids were extracted, each plasmid was double-digested with restriction enzymes SmaI and KprI, the reaction mixture was separated by 0.7% normal agarose gel electrophoresis, and a plasmid containing an insert of about 1.0kb was selected (not necessarily identified separately) to give plasmid pTN. Can be stored and amplified for later use.

The above process is illustrated in fig. 6.

EXAMPLE 5 construction of plasmid pGN a preparation of plasmid vector and NF-IL6 Gene fragment

1. Plasmid pMSG (Pharmacia Biotech, containing mouse mammary tumor virus [ MMTV ] promoter) 0.5. mu.g (0.5. mu.l), 10 Xrestriction enzyme buffer A (Boehringer-Mannheim, Inc.) 2. mu.l, sterilized water 17. mu.l and restriction enzyme SmaI 1. mu.l were taken, mixed, incubated at 25 ℃ for 2 hours for enzyme reaction, then restriction enzyme XhoI 3. mu.l was added, and incubation at 37 ℃ was continued for 2 hours. The reaction solution was extracted 1 time with phenol/chloroform, once with chloroform, and the plasmid was precipitated with ethanol, dissolved in 10. mu.l of 10m mol/L Tris.HCl pH8.0-1m mol/L EDTA, as solution A.

2. Plasmid pBlue 6100.5. mu.g (0.5. mu.l), 10 Xrestriction endonuclease buffer A2. mu.l, sterilized water 17. mu.l and restriction endonuclease SmaI 1. mu.l were mixed, incubated at 25 ℃ for 2 hours to effect enzymatic reaction, then restriction endonuclease XhoI 3. mu.l was added, and incubation at 37 ℃ was continued for 2 hours. The reaction mixture was separated by 0.7% low melting point agarose gel electrophoresis, the 1.0kb band was excised, and plasmid DNA was extracted from the gel and dissolved in 10. mu.l of 10mmol/L Tris.HCl pH8.0-1mmol/L EDTA as solution B, i.e., the NF-IL6 coding region. Ligation of the NF-IL6 coding region with the plasmid vector pMSG and bacterial transformation

Mu.l of solution A, 10. mu.l of solution B, 2. mu.l of 10XT4 DNA ligase buffer, 2. mu.l of 10m mol/L ATP and 1. mu.l of T4 DNA ligase were mixed and incubated at 16 ℃ overnight for enzymatic reaction. The next day, 1. mu.l of the reaction mixture was used to transform E.coli DH 5. alpha. and spread on LB solid medium containing 100. mu.g/ml ampicillin, overnight, colonies were randomly picked up, 1ml of LB liquid medium was used to amplify the bacteria, plasmids were extracted, each plasmid was double-digested with restriction enzymes SmaI and XhoI, the reaction mixture was separated by 0.7% normal agarose gel electrophoresis, and a plasmid exhibiting an insert of about 1.0kb (not necessarily identified) was selected as plasmid pGN. Can be stored and amplified for later use.

The above process is illustrated in fig. 7.

EXAMPLE 6 construction of Linear plasmid pVN

Mu.g (100. mu.l) of the plasmid pVN 100 constructed in example 3 was added to 20. mu.l of a 10 Xrestriction enzyme PvuI buffer, 70. mu.l of sterilized water and 10. mu.l (100 units) of restriction enzyme Pvul, and the mixture was mixed and incubated at 37 ℃ for 2 hours to carry out an enzymatic reaction. The reaction solution is extracted once by phenol/chloroform and once by chloroform, the plasmid is precipitated by ethanol and dissolved in 100 mul of 10m mol/L Tris.HCl pH8.0-1m mol/L EDTA, and then the linear plasmid pVN is obtained. Can be stored for later use.

Example 7: experiment for treating human liver cancer nude mouse tumor by plasmid pSN

Nude mice were inoculated with 15 mice subcutaneously at 10 sites per injection⁷Human liver cancer SMMC7721 cells. After 1 week, tumors were formed. Each group of 4 patients (3 control groups without any drug) was injected with the following substances at the tumor site:

0.1 group … … … … … pSN 0.1. mu.g plus liposomes 50. mu.g

Group 1 … … … … … pSN 1. mu.g plus liposome 50. mu.l

10 groups … … … … … pSN 10. mu.g plus Liposome 50. mu.l control group … … … … … 0

After injection, the major (a) and minor (b) diameters of each tumor were measured every 3 days, and the tumor volume V was calculated according to the following formula:

V=0.4ab². The results showed that all the tumors injected with pSN stopped proliferating and tended to shrink, and part of the tumors finally disappeared. As shown in table 1 and fig. 8.

Table 1 treatment of plasmid pSN on human hepatoma nude mice therapy experiment day 0.1 post injection, group 1, group 10, control group 0111130.49 + -0.410.53 + -0.110.66 + -0.100.94 + -0.2160.21 + -0.200.21 + -0.080.24 + -0.141.7 + -0.5690.06 + -0.040.11 + -0.080.04 + -0.051.9 + -0.67 data in table is the ratio of the tumor volume observed day to the tumor volume at the time of injection (mean + -standard deviation), and the data for this implementation is plotted in graph 8.

EXAMPLE 8 experiment on treatment of tumor implanted in nude mouse with human gastric cancer by plasmid pSN (1)

12 nude mice were injected intraperitoneally with 6X 10 injections⁶In 8 of the individual gastric cancer cells MKN-45, 10. mu.g of pSN plus liposome was intraperitoneally injected 50. mu.l and 250. mu.l of physiological saline one week later, and the remaining 4 cells were controls. After 8 days, the results were as follows:

a)4 control mice are all in an moribund state, the abdomen is swollen, and a large amount of intra-abdominal bleeding is found by dissection (see figure 9);

b) of 12 treated mice, 5 mice were also in an moribund state, with swollen abdomen, large tumors found by dissection and massive intra-abdominal bleeding; 4, the tumor and internal bleeding are found in dissection, but the degree is obviously lighter than that of the control; 3 had no difference with normal mice, no tumor was found by dissection, indicating that the disease was cured (see FIG. 10). Therefore, the injection of pSN can obviously slow down the proliferation speed of the tumor and even cure the tumor.

EXAMPLE 9 experiment on treatment of tumor implanted in human Lung cancer nude mouse with plasmid pSN (2)

Nude mice were 6 mice, each injected subcutaneously 10 times⁷One week later, tumors were formed in the individual lung cancer SPC-A-1 cells. Tumor-bearing mice were divided into 3 groups on average, 1 group was a control, and the remaining 2 groups were injected with psn0.1 μ g and 1 μ g, respectively, at the tumor site. The major axis (a) and minor axis (b) of each tumor were measured every 2-3 days, and tumor volume V was calculated according to the following formula:

V=0.4ab². The results showed that the proliferation rate of the pSN injected tumors was significantly slower than the control, as shown in table 2 and fig. 11.

TABLE 2 plasmid pSN treatment experiment on human Lung cancer plantama days after injection 0.1 group 1 group control group

0 1 1 1

4 2.19+-1.11 0.80+-0,48 3.53+-1.11

6 2.15+-0.16 1.40+-0.58 6.18+-1.63

8 6.45+-3.45 2.63+-0.76 9.78+-4.47

129.38 + -3.765.48 + -1.7416.2 + -6.44 the data in the table are the ratio of the observed daily tumor volume to the tumor volume at the time of injection (mean + -standard deviation), and the experimental data are plotted in FIG. 11.

Claims

1. A series of human interleukin 6 nuclear transcription factor expression plasmids, characterized in that it has the following basic structure:

a circular double-stranded DNA molecule formed by sequentially connecting a promoter, an NF-IL6 coding region, a DNA sequence 1, a polyA signal and a DNA sequence 2, wherein,

promoter = the promoter of any eukaryotic gene, which is an element that promotes the expression of the gene immediately downstream thereof;

DNA sequence 1= DNA sequence of any eukaryotic plasmid vector between the multiple cloning site and the polyA signal, containing splicing signals for mRNA (messenger ribonucleic acid);

DNA sequence 2= DNA sequence of any eukaryotic plasmid vector between the polyA signal and the promoter, contains the replication initiation site of the plasmid vector DNA, and may also contain antibiotic resistance genes, including genes that confer resistance to a certain antibiotic to bacteria or to eukaryotic cells.

2. A series of human interleukin 6 nuclear transcription factor expression plasmids, characterized in that it has the following basic structure:

a linear double-stranded DNA molecule formed by sequentially connecting a DNA sequence 2 (one part), a promoter, an NF-IL6 coding region, a DNA sequence 1, a polyA signal and the DNA sequence 2 (the other part); wherein,

DNA sequence 2= DNA sequence of any eukaryotic plasmid vector downstream of the polyA signal and/or upstream of the promoter, containing the replication initiation site of the plasmid vector DNA, and may also contain antibiotic resistance genes, including genes that confer resistance to a certain antibiotic to bacteria or to eukaryotic cells.

3. The human interleukin 6 nuclear transcription factor expression plasmid series of claims 1 and 2, wherein each plasmid structure in the expression plasmid series comprises any one of the following promoters: the SV40 virus early promoter; [ II ] SV40 virus late promoter; [ III ] cytomegalovirus promoter; [ IV ] a promoter of metallothionein gene; [ V ] a promoter of myosin gene; [ VI ] an adenovirus promoter; [ VII ] vaccinia virus promoter; (viii) a herpes virus promoter; [ IX ] influenza virus promoter; a promoter of [ X ] actin gene; [ XI ] a promoter of the collagen gene; [ XII ] papilloma virus promoter; [ XIII ] polyoma virus promoter; a promoter of the [ XIV ] alpha-fetoprotein gene; (ii) a promoter of [ XV ] carcinoembryonic antigen gene; the [ XVI ] Mouse Mammary Tumor Virus (MMTV) promoter.

4. The use of the plasmid series for the expression of human interleukin 6 nuclear transcription factor as claimed in claims 1 and 2, characterized in that the plasmid series can be used as gene medicine for the treatment and prevention of various tumors.