阅读说明：本技术 一种过表达vault RNA 2-1基因质粒构建方法及其应用 (Construction method and application of plasmid for over-expressing vault RNA2-1 gene ) 是由 马祥雪 于 2020-04-03 设计创作，主要内容包括：发明公开了一种针对vault RNA 2-1基因过表达重组质粒的构建方法及其在基因功能研究方面的应用。本发明涉及一种vault RNA 2-1基因,并提供vault RNA 2-1基因过表达重组真核表达质粒pcDNA3.1(+)-vtrna2-1构建方法,以及在人结肠癌细胞中vault RNA 2-1基因研究的实验方法。(The invention discloses a construction method of a vault RNA2-1 gene overexpression recombinant plasmid and application thereof in gene function research. The invention relates to a vault RNA2-1 gene, and provides a construction method of a vault RNA2-1 gene overexpression recombinant eukaryotic expression plasmid pcDNA3.1(+) -vtrna2-1, and an experimental method for study of the vault RNA2-1 gene in a human colon cancer cell.)

1. a eukaryotic recombinant expression plasmid, characterized by: the target sequence of the DNA has the deoxynucleotide sequence as follows:

CGGGTCGGAGTTAGCTCAAGCGGTTACCTCCTCATGCCGGACTTTCTATCTGTCCATCTCTGTGCTGGGGTTCGAGACCCGCGGGTGCTTACTGACCCTTTTATGCAA。

2. the eukaryotic recombinant expression plasmid of claim 1, wherein the eukaryotic recombinant expression plasmid is constructed by recombining a vtrna2-1 gene fragment with the eukaryotic expression plasmid.

3. The method for constructing a eukaryotic recombinant plasmid according to claim 1, comprising:

A) synthesizing a human vtrna2-1 gene fragment by RT-PCR;

B) the vtrna2-1 gene fragment and TA cloning vector2.1 ligation, the recombinant plasmid2.1-vtrna2-1 to DH5 α competent cells, identifying and screening positive clones by cloning PCR, extracting positive cloning plasmids, carrying out enzyme cutting by using restriction enzymes, wherein the enzyme cutting sites are HindIII and XhoI respectively,

C) the enzyme digestion product is connected with a eukaryotic plasmid vector pcDNA3.1(+), the connection product is transformed into a DH5 alpha competent cell, the identification is carried out through cloning PCR, positive clone is identified and screened, plasmid is extracted, eukaryotic recombinant plasmid pcDNA3.1(+) -vtrna2-1 is obtained,

D) the eukaryotic recombinant plasmid pcDNA3.1(+) -vtrna2-1 is identified by using a restriction enzyme cutting map.

4. Use of the eukaryotic recombinant expression plasmid according to any one of claims 1 to 2 for the preparation of a medicament for inhibiting proliferation of colon cancer cells Caco-2 cells.

The technical field is as follows:

the invention relates to a construction method and application of eukaryotic recombinant expression plasmid for over-expressing vault RNA 2-1.

Technical Field

Colorectal cancer (CRC) is one of common malignant tumors of the digestive tract, the incidence rate of which is 3 rd of malignant tumors worldwide, the mortality rate of which is 2 nd of malignant tumors worldwide, and the CRC is the first digestive system malignant tumor to cause diseases and death worldwide, and seriously threatens life and health. In recent years, with westernization of life style and dietary structure in China, the incidence of colorectal cancer generally shows an increasing trend, and the colorectal cancer becomes malignant tumors of the 2 nd and 1 st incidence of digestive system diseases in China. New cases of colorectal cancer in China exceed 52.1 ten thousand, death cases are about 24.8 ten thousand, the new cases and the death cases are both close to 30% of the colorectal cancer cases in the same period all over the world, and the disease burden is heavy. At present, the 5-year survival rate of colorectal cancer in China is far lower than that of American and Japanese Korean, more than 85 percent of colorectal cancer is found to be in the late stage, and even through comprehensive treatment such as operation, radiotherapy and chemotherapy, targeted treatment and the like, the 5-year survival rate of patients is still obviously lower than 40 percent; in contrast, the 5-year survival rate after early colorectal cancer treatment can exceed 95% and even be completely cured. Therefore, early diagnosis and early treatment of CRC are particularly important. At present, the molecular mechanism of early colorectal carcinoma is not completely elucidated, so that the deep understanding of the molecular mechanism of colorectal carcinoma plays an important role in the research and development of early diagnosis and treatment of cancer.

Genetic engineering, also known as gene recombination technology, is a technology developed in the seventies of the twentieth century to operate DNA in vitro, and is widely used for basic research of diseases, gene therapy, gene immunization, gene vaccines and the like, and the three elements of the technology are respectively: enzyme, target gene and vector. The eukaryotic expression vector is a vector used for constructing an eukaryotic gene expression system in genetic engineering. In recent decades, various researchers around eukaryotic expression vectors have made various diseases, genes and other related researches, have made a lot of work in the aspect of genetic engineering, and have opened up a new way for medical science research.

Non-coding RNA is a key molecule for the development of tumorigenesis that has emerged in recent years, and its clinical value is receiving close attention. Vault RNA (vtrna) is a eukaryotic non-coding RNA of 84-141nt in length, which is transcribed by RNA polymerase III. They are associated with conserved vault proteins that form vault particles, a complex structure whose function and relevance in cancer is not known to date. The human vtrna family comprises four subtypes, vtrna1-1, vtrna1-2, vtrna1-3 and vtrna 2-1. Different evidence suggests that epigenetic control of vtrna2-1 is complex and may be of clinical significance. Independent reports of breast, lung, colon, bladder, esophageal, and gastric cancers show that the promoters are methylated to a different extent in tumor tissues than in normal tissues.

Disclosure of Invention

The invention uses gene recombination technology to construct a recombinant eukaryotic expression vector pcDNA3.1(+) -vtrna2-1 aiming at the gene vtrna2-1, transfects epithelial cells of human colon cancer with the recombinant eukaryotic expression vector pcDNA3.1(+) -vtrna2-1, constructs a cell strain of the gene vtrna2-1, provides a good tool for researching the function of the gene vtrna2-1, lays a foundation for further researching the biological function of the gene vtrna2-1 in colorectal cancer, and provides an effective experimental method for researching the function of the gene vtrna2-1 in the human colorectal cancer or other malignant tumors.

According to one aspect of the invention, the inventor provides a novel eukaryotic recombinant plasmid and a preparation method thereof, and proves that the recombinant plasmid is a good tool for researching the function of the vtrna2-1 gene and provides an effective experimental method for researching the function of the vtrna2-1 gene of human colorectal cancer.

The vtrna2-1 gene is registered in GenBank under the registration number NG-051612.1, and is positioned at 5q31.1 and has a total length of 108 bp. The preparation method of the eukaryotic recombinant plasmid sequentially comprises the following steps:

A) synthesizing a human vtrna2-1 gene fragment by RT-PCR;

B) connecting the obtained nucleotide fragment with eukaryotic plasmid, wherein the restriction enzyme sites of the insert fragment are HindIII and XhoI respectively, transforming the eukaryotic recombinant plasmid into competent bacteria for culture, and screening eukaryotic recombinant plasmid clone with correct insertion;

C) extracting eukaryotic recombinant plasmid, and analyzing the constructed eukaryotic recombinant plasmid by using restriction enzyme digestion map.

The recombinant plasmid obtained above was transfected into human colon cancer cells Caco-2 cells and HCT-116 cells to observe the cell biological properties. The result shows that when the recombinant plasmid is transfected into Caco-2 cells and HCT-116 cells of human colon cancer cells, the vtrna2-1 gene has the function of inhibiting cell proliferation. Therefore, the invention is helpful for researching the function of the vtrna2-1 gene and provides a molecular biological tool for researching the effect of the vtrna2-1 gene on the pathogenesis of human colorectal cancer.

According to yet another aspect of the present invention, there is provided a eukaryotic recombinant expression plasmid characterized in that: the target sequence of the DNA has the deoxynucleotide sequence as follows:

CGGGTCGGAGTTAGCTCAAGCGGTTACCTCCTCATGCCGGACTTTCTATCTGTCCATCTCTGTGCTGGGGTTCGAGACCCGCGGGTGCTTACTGACCCTTTTATGCAA。

according to a further aspect of the invention, the eukaryotic recombinant expression plasmid is constructed by recombining the vtrna2-1 gene fragment with the eukaryotic expression plasmid.

According to still another aspect of the present invention, there is provided a method for preparing the above eukaryotic recombinant plasmid, characterized by comprising:

A) synthesizing a human vtrna2-1 gene fragment by RT-PCR;

B) the vtrna2-1 gene fragment and TA cloning vector2.1 connection, transforming the recombinant plasmid pCR 2.1-vtrna2-1 into DH5 α competent cells, identifying and screening positive clones by cloning PCR, extracting positive cloning plasmids, carrying out enzyme digestion by using restriction enzymes, wherein the enzyme digestion sites are HindIII and XhoI respectively,

C) the enzyme digestion product is connected with a eukaryotic plasmid vector pcDNA3.1(+), the connection product is transformed into a DH5 alpha competent cell, the identification is carried out through cloning PCR, positive clone is identified and screened, plasmid is extracted, eukaryotic recombinant plasmid pcDNA3.1(+) -vtrna2-1 is obtained,

D) the eukaryotic recombinant plasmid pcDNA3.1(+) -vtrna2-1 is identified by using a restriction enzyme cutting map.

According to another aspect of the invention, the application of the eukaryotic recombinant expression plasmid in preparing a medicament for inhibiting the proliferation of colon cancer cells Caco-2 cells is provided.

Drawings

FIG. 1 is a schematic structural diagram of eukaryotic recombinant vector pcDNA3.1(+) according to the present invention.

FIG. 2 is a schematic structural diagram of a eukaryotic recombinant plasmid vector pcDNA3.1(+) -vtrna2-1 according to the present invention.

FIG. 3 is a graph showing a band diagram of the PCR amplification product of vtrna2-1 according to the present invention.

FIG. 4 is the restriction enzyme identification map of eukaryotic recombinant plasmid vector pcDNA3.1(+) -vtrna2-1 of the present invention.

FIG. 5 is a histogram of the transfection efficiency of the eukaryotic recombinant plasmid vector pcDNA3.1(+) -vtrna2-1 of the present invention.

FIG. 6 is a diagram showing the proliferation of human colon cancer cell Caco-2 cell line after the eukaryotic recombinant plasmid vector pcDNA3.1(+) -vtrna2-1 is transfected.

FIG. 7 is the cell growth curve of human colon cancer cell Caco-2 after the eukaryotic recombinant plasmid vector pcDNA3.1(+) -vtrna2-1 is transfected.

FIGS. 8A and 8B show the expression of Caco-2 proliferating cell nuclear antigen PCNA after the eukaryotic recombinant plasmid vector pcDNA3.1(+) -vtrna2-1 is transfected.

FIGS. 9A-9C show the expression of Caco-25-bromodeoxyuridine Brdu in human colon cancer cells transfected by the eukaryotic recombinant plasmid vector pcDNA3.1(+) -vtrna2-1 of the present invention and its control.

FIGS. 10A-10C show the expression of the eukaryotic recombinant plasmid vector pcDNA3.1(+) -vtrna2-1 transfected human colon cancer cell CaCo-2 nuclear antigen Ki67 and its control.

The specific implementation process comprises the following steps:

the present invention will be further described with reference to specific examples.