CN108070589A - Nucleic acid molecules CTL4HSH9, its preparation method and application - Google Patents

Abstract

The invention belongs to the fields of cell biology and medicine. The present invention provides a nucleic acid molecule, which has the activity of inhibiting the expression of CTL4 gene in vitro; and the inhibited target element includes the sequence shown in SEQ ID NO 1 . The present invention provides a recombinant vector containing the nucleic acid molecule, and also provides a kit for inhibiting the expression of CTL4 gene in vitro, which includes the above-mentioned nucleic acid molecule; the sequence of the nucleic acid molecule is as shown in SEQ ID NO 2 or SEQ ID NO 3 shown. The invention also provides the preparation method and application of the nucleic acid.

Description

Nucleic acid molecule CTL4HSH9, its preparation method and application

technical field

The invention belongs to the field of cell biology and medicine, in particular, the invention relates to a nucleic acid molecule and its preparation method and application.

Background technique

Tumor is an abnormal lesion formed by the body under the action of various carcinogenic factors when a certain cell in a local tissue loses its normal regulation of its growth at the gene level, resulting in its clonal abnormal proliferation. The number of cancer cases in my country is quite large, accounting for 55% of the total number of cases in the world. Tumor disease has now risen to become the world's No. 2 "killer", and its death toll is second only to cardiovascular disease.

Benign tumors have less impact on the body, mainly showing symptoms of local compression and obstruction. Malignant tumors have a serious impact on the body due to their immature differentiation, rapid growth, infiltration and damage to the structure and function of organs, and the possibility of metastasis. In addition to local compression and obstruction symptoms similar to those of benign tumors mentioned above, malignant tumors may also cause fever and intractable pain, and severe emaciation, fatigue, anemia, and systemic exhaustion may appear in the advanced stage.

In recent years, foreign medical circles have gained a new understanding of the pathogenesis of tumor diseases on the basis of cells. Based on the further understanding of the pathogenesis of tumors, people use various approaches to research and develop drugs that can specifically and effectively kill tumor cells without toxicity to normal cells. At present, chemotherapy and radiotherapy are still the first choice for the treatment of cancer. Although the two have achieved considerable curative effect on the treatment of tumors, they have relatively large toxic and side effects due to the lack of specificity to tumor cells, and some tumor cells have a certain effect on chemotherapy. And radiotherapy treatment insensitivity, thus limiting their clinical application to a large extent. In recent years, in order to develop drugs that can specifically kill cancer cells and have no toxic and negative effects on normal cells, people have paid great attention to and invested heavily in research on the pathogenesis of cancer from the cellular and molecular levels. For example, "colorectal cancer" (CRC) has always been known as "rich man's disease" in tumors, which shows that it is closely related to people's living habits. In Shanghai, with the improvement of living standards, the incidence of colorectal cancer has jumped to the second place among common malignant tumors, second only to lung cancer. According to statistics from the Cancer Hospital Affiliated to Fudan University, the number of colorectal cancer operations has increased year after year in recent years. In 2007, the number of colorectal cancer operations in the hospital was nearly 900 cases. It has always been a hot spot in this field to develop drugs or compounds that can specifically kill tumor cells and have no toxic side effects on normal cells.

Contents of the invention

The technical problem to be solved by the present invention is to provide a nucleic acid molecule for inhibiting the expression of CTL4 gene in vitro. By preparing and using the nucleic acid molecule to inhibit the expression of the CTL4 gene in vitro, the effect of maintaining the normal physiological state of the target cell is achieved.

The invention provides a new nucleic acid molecule, which has the activity of inhibiting the expression of CTL4 gene in vitro;

and

Its inhibitory target element includes the sequence shown in SEQ ID NO 1 .

More preferably, the target element has the sequence shown in SEQ ID NO 1 .

The present invention also provides a recombinant vector comprising the above-mentioned nucleic acid molecule.

Wherein, A, T, G and C are adenine nucleotides, thymine nucleotides, guanine nucleotides and cytosine nucleotides, respectively.

In the present invention, the term "CTL4HSH9" refers to a nucleic acid molecule that has the activity of inhibiting the expression of vascular CTL4, and the inhibitory target element includes the sequence shown in SEQ ID NO 1 . The term also includes nucleic acid molecules whose sequence of the element of interest is at least 90% homologous to SEQ ID NO 1 .

The term also includes the addition of several (usually within 5, preferably within 3) nucleotides at the 5' and/or 3' end of the target element, or after adding several dT (deoxythymidine) at the 3' end formed sequence.

In the present invention, various vectors known in the art, such as commercially available vectors, can be used. For example, a commercially available vector is selected, and then the DNA sequence corresponding to the CTL4HSH9 of the present invention is operably linked to the expression control sequence to form a recombinant vector. For example, lentivirus, adenovirus, or forest encephalitis virus expression system (Semliki ForestVirus) can be used. Lentivirus belongs to the subgenus of retrovirus, represented by human immunodeficiency virus (HIV). Lentiviruses not only have the ability to infect dividing target cells and integrate into their genomes, but especially have the ability to infect a variety of non-dividing cells including neuron cells, macrophages, liver cells, cardiomyocytes and stem cells. Therefore, lentivirus vectors As an effective tool for gene transfer, it has been widely used in the study of gene function, especially gene therapy.

In another aspect, the present invention provides a kit for inhibiting the expression of CTL4 gene in vitro, which includes the above nucleic acid molecule.

Preferably, the sequence of the nucleic acid molecule is shown as SEQ ID NO 2 or SEQ ID NO 3.

For example, it has a nucleic acid molecule whose sequence is shown in SEQ ID NO 2.

Alternatively, it has a nucleic acid molecule whose sequence is shown in SEQ ID NO 3.

More preferably, it has the nucleic acid molecules whose sequences are shown in SEQ ID NO 2 and SEQ ID NO 3.

The present invention also provides a method for preparing the above kit, including the step of preparing a nucleic acid molecule whose sequence is shown in SEQ ID NO 2 or SEQ ID NO 3: the nucleic acid molecule shown in SEQ ID NO 2 or SEQ ID NO 3 according to its sequence Artificial synthesis; that is, according to the nucleic acid sequence, each ribonucleic acid molecule is sequentially dehydrated and condensed.

Alternatively, enzyme cleavage is used, and each functional element is prepared separately and then connected.

In the third aspect, the present invention provides a method for inhibiting the expression of CTL4 gene in in vitro cells, that is, the nucleic acid molecules are transfected into in vitro cells through an appropriate medium.

For example, adding to the culture medium of in vitro cells, or transfecting target cells with vectors, including viral vector systems.

Specifically, the method can include:

(1) Amplify nucleic acid molecules;

(2) Construction of expression vectors containing nucleic acid molecules;

(3) Transform the expression vector obtained in step (2) into target in vitro cells.

The present invention also provides the application of the above-mentioned nucleotide molecule in the preparation of a medicament for inhibiting proliferation of cells in vitro, especially tumor cells. Experimental results show that the CTL4HSH9 of the present invention can significantly inhibit the expression of CTL4. Experiments have proved that CTL4 (NCBI gene ID80736) has important biological functions of the immune system, but in some cases, it can also cause damage to the body, causing allergic diseases or immune diseases. Inhibiting the expression of CTL4 in a limited range of cells can effectively control the excessive reaction caused by it and maintain the normal physiological activities of the body.

The nucleic acid molecules of the present invention and their analogs, when administered (administered) therapeutically, can provide various effects. Generally, these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably about 6-8, although the pH value can be changed according to the Depending on the nature of the substance formulated and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intramuscular, intraperitoneal, subcutaneous, intradermal, or topical administration.

Taking the nucleic acid molecule of the present invention as an example, it can be combined with a suitable pharmaceutically acceptable carrier. Such pharmaceutical compositions contain a therapeutically effective amount of the compound and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should match the mode of administration. The nucleic acid molecules of the present invention can be prepared in the form of injections, for example, by conventional methods using physiological saline or aqueous solutions containing glucose and other adjuvants. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions. The active ingredient is administered in a therapeutically effective amount, for example about 1 microgram/kg body weight to about 10 mg/kg body weight per day. In addition, the invention can also be used with other therapeutic agents.

When the nucleic acid molecule of the invention is used as a medicament, a therapeutically effective dose of the polypeptide can be administered to a mammal, wherein the therapeutically effective dose is usually at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 8 milligrams /kg body weight, preferably the dosage is about 10 μg/kg body weight to about 1 mg/kg body weight. Of course, factors such as the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians. In the present invention, the medicament is a pharmaceutical composition consisting of therapeutically effective nucleic acid molecules and pharmaceutical carriers or excipients.

The present invention provides a nucleic acid molecule, which has the activity of inhibiting the expression of CTL4 gene in vitro; and the inhibited target element includes the sequence shown in SEQ ID NO 1 . The present invention provides a recombinant vector containing the nucleic acid molecule, and also provides a kit for inhibiting the expression of CTL4 gene in vitro, which includes the above-mentioned nucleic acid molecule; the sequence of the nucleic acid molecule is as shown in SEQ ID NO 2 or SEQ ID NO 3 shown. The invention also provides the nucleic acid and its preparation method and application. Experiments show that the nucleic acid molecule of the present invention can inhibit at least 45% of the expression of CTL4 gene in vitro.

Detailed ways

Example 1 Design and screening of CTL4 inhibitory molecules

Using GATGATGTCTACCATGTTCTA (SED ID NO 1) in the CTL4 sequence as a target to screen inhibitory molecules, the sequence is 5'-ACCTCGATGATGTCTACCATGTTCTATCAAGAGTAGAACATGGTAGACATCATCTT-3' (SED ID NO2) or 5'-CAAAAAAGATGATGTCTACCATGTTCTACTCTTGATAGAACATGGTAGACATCATCG-3' (SED IDNO CTL) candidate nucleic acid molecule as Inhibitor, called CTL4HSH9.

293T cells were planted in 24-well plates with a plate density of 2*10 ⁴ /well.

24h (hour), 72h, and 120h after seeding the plate, the CTL4HSH9 fragment was repeatedly transfected, and dissolved in Opti-MEM (Opti-MEM IReduced Serum Medium, Invitrogen Company, 31985-070) as a solvent to make the final concentration reach 40nM. Transfection reagent used lipofectamin2000 (11668-027, Invitrogene Company).

The cells were digested at 24h, 72h, and 120h after the first transfection, and some (5*10 ⁴ ) cell samples were collected at 72h and 120h. For Western detection, the antibody used Nogo (N-18) SC-11027 (Santa Cruz Company) CTL4 endogenous protein expression level changes.

Conclusion: Compared with the siRNA-negative fragment NS (target replaced by GAATTCCGGTCGCATTATTAT, SEQ ID NO 4), 5'-ACCTCGATGATGTCTACCATGTTCTATCAAGAGTAGAACATGGTAGACATCATCTT-3' or 5'-CAAAAAAGATGATGTCTACCATGTTCTACTCTTGATAGAACATGGTAGACATCATCG-3' can be seen at least 72 hours after transfection to reduce the expression of CTL4 protein by 50%. This shows that the CTL4HSH9 of the present invention can obviously inhibit CTL4.

Example 2 Cell-level screening experiments for the reduction effect on CTL4 gene expression

Experimental steps:

293T cells (purchased from the Cell Bank of Chinese Academy of Sciences) were planted in 96-well plates with a plate density of 0.8*10 ⁴ /well.

After 24 hours, the lentivirus (lentivirus, Jikai Company) carrying CTL4HSH9 was transfected into the cells. The titer of Lentivirus is 106TU/ul, and the MOI is 100, that is, 80ul of virus solution is added to each well. The specific transduction method is as follows:

Replace each well of cells with 50ul of fresh medium (pH7.0), add an appropriate amount of virus solution, and add 50ul of medium to each well after 7-8 hours. Change the medium after 24h. 48-72h observed GFP (green fluorescence) fluorescence, detection of transduction efficiency.

The cells were expanded and cultured, and the cells were sorted by GFP green fluorescence by flow cytometry, and the positive cells for virus transduction were screened out, and the positive rate was detected.

The changes in the expression level of CTL4 endogenous protein in positive cells and negative cells were detected by Western analysis.

in conclusion:

A. The GFP fluorescence was observed for 48 hours, and the positive rate was about 50%, indicating that the method of virus transduction experiment was correct.

B. Sorting transduced cells by flow cytometry, the transduction rate of positive group and negative group were both greater than 90% (data displayed by flow cytometry).

C. The expression level of CTL4 protein in positive cells by Western assay was significantly lower than that in negative cells.

SEQUENCE LISTING

<110> Fudan University

<120> Nucleic acid molecule CTL4HSH9, its preparation method and application

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Claims (10)

1. A nucleic acid molecule, characterized in that it has the activity of inhibiting the expression of CTL4 gene in vitro; and Its inhibitory target element includes the sequence shown in SEQ ID NO 1 . 2. A nucleic acid molecule according to claim 1, wherein the target element has the sequence shown in SEQ ID NO 1 . 3. A recombinant vector, characterized in that it comprises the nucleic acid molecule as claimed in claim 1. 4. A test kit for inhibiting CTL4 gene expression in vitro, characterized in that it comprises the nucleic acid molecule according to claim 1; The sequence of the nucleic acid molecule is shown in SEQ ID NO 2 or SEQ ID NO 3. 5. A kit as claimed in claim 4, characterized in that it has a nucleic acid molecule whose sequence is shown in SEQ ID NO 2. 6. A kit as claimed in claim 4, characterized in that it has a nucleic acid molecule whose sequence is shown in SEQ ID NO 3. 7. A kit as claimed in claim 4, characterized in that it has nucleic acid molecules whose sequences are shown in SEQ ID NO 2 and SEQ ID NO 3. 8. The preparation method of any kit in claim 4-7, characterized in that, said preparation method comprises the step of preparing a nucleic acid molecule whose sequence is shown in SEQ ID NO 2 or SEQ ID NO 3: such as SEQ ID The nucleic acid molecule shown in NO 2 or SEQ ID NO 3 is artificially synthesized according to its sequence. 9. A method for inhibiting the expression of CTL4 gene in in vitro cells, characterized in that the nucleic acid molecule according to claim 1 is transfected into in vitro cells through an appropriate medium. 10. The application of the nucleic acid molecule according to claim 1, characterized in that it comprises the application of the nucleic acid molecule in preparing and inhibiting the expression of CTL4 in vitro; Said application includes the following steps: (1) amplifying the nucleic acid molecules in the kit according to claim 4; (2) Constructing an expression vector containing the nucleic acid molecule described in the kit of claim 4; (3) Transform the expression vector obtained in step (2) into target in vitro cells.