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Application of cell cycle regulatory gene fam114a2

A cell cycle and gene regulation technology, applied in the field of molecular biology, can solve the problems of new genes added in the wrong place, failure to reach, recurrence, etc., and achieve the effect of enhancing cell proliferation vitality

Active Publication Date: 2021-01-26
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When genes are added to the DNA, there is also the risk of new genes being added in the wrong place and causing other damage
In addition, to cure tumors, every tumor cell must be subjected to gene therapy, otherwise there is a possibility of recurrence, but the current gene transfer technology cannot meet this requirement

Method used

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  • Application of cell cycle regulatory gene fam114a2
  • Application of cell cycle regulatory gene fam114a2
  • Application of cell cycle regulatory gene fam114a2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1, Bmfam114a2 gene acquisition and analysis

[0034] The Bmfam114a2 gene comes from the positional cloning of the short-bodied silkworm (Squab, Sq) mutant in the Silkworm Gene Bank of Southwest University. The possibility of other genes in the region as candidate genes for short-bodied silkworms, and finally 4 genes in the abnormal region of chromosome fragments were included in short-bodied silkworm candidate genes for research, one of which was Bmfam114a2, and then the complete Bmfam114a2 gene was obtained from the silkworm genome Sequence, Bmfam114a2 gene length is about 12.3kb, ORF box is 1731bp (SEQID NO.1), consists of 10 exons, encodes 576 amino acids, and encodes a protein with a molecular weight of 62.2kDa ( figure 1 ). Sequence alignment and domain analysis showed that Bmfam114a2 protein had a DUF719 (DUF means Domain of unknown function) domain. The structural domain of this gene is highly conserved in different species, but its specific function is...

Embodiment 2

[0040] Example 2, construction of fusion tag expression plasmid and functional analysis of Bmfam114a2 protein

[0041] Primers were designed according to the full-length ORF of Bmfam114a2 obtained by cloning, and the specific primers were as follows:

[0042] 5'-tgctctaga atggat tacaaggatgacgacgataagatggctacaagtgatagtgaa-3' (SEQID NO.4), the underline represents the Xba I restriction site;

[0043] 5'-ccg ctcgag ttacttatcgtcgtcatccttgtaatccacggcacctatttgta-3' (SEQ ID NO.5), the underline represents the Xho I restriction site;

[0044] Then use the pMD19-T vector containing the Bmfam114a2 gene ORF as a template, and the sequences shown in SEQ ID NO.4 and SEQ ID NO.5 as primers for PCR amplification, and the amplified product is double digested with Xba I and Xho I, and then ligated to the PIZ / V5-dsRed vector. After transformation, single-clonal plaques were selected, verified by electrophoresis of bacterial liquid, PCR of bacterial liquid and double enzyme digestion, and ...

Embodiment 3

[0047] Embodiment 3, construction interference plasmid and overexpression plasmid and transfection

[0048] According to the protein with potential function obtained by co-immunoprecipitation technique, and the result of functional analysis of fam114a2, the Bmfam114a2 insect interference vector and the Bmfam114a2 overexpression vector were constructed.

[0049] Construction of interference vectors: with the online analysis tool BLOCK-iT TM RNAi Designer predicted the siRNA sequence of the candidate gene and compared it in the silkworm genome database to exclude the siRNA sequence with non-specific binding ability. As a result, the fam114a2 gene interference target sequence was obtained, and the specific sequences were as follows:

[0050] Target sequence 1: 5'-tagagatgctatcccggcaagtgaa-3' (SEQ ID NO.6);

[0051] Target sequence 2: 5'-cagagaatacggagcaaatacataa-3' (SEQ ID NO.7);

[0052] Synthesize the fam114a2 interference fragment according to the target sequence, its nuclei...

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Abstract

The invention discloses the application of the cell cycle regulating gene fam114a2. It is found through research that the gene has a cell cycle regulating effect and inhibits cell proliferation, so it can be used as a cell cycle regulating agent and as a target gene for tumor treatment; and the gene is used in renal cancer , liver cancer, gastric cancer and paracancerous tissues are specifically highly expressed, so it can also be used as a diagnostic marker for renal cancer, liver cancer, and gastric cancer, and is of great significance to the diagnosis and treatment of tumors.

Description

technical field [0001] The invention belongs to the field of molecular biology, and specifically relates to the application of the cell cycle regulating gene fam114a2. Background technique [0002] Tumor is defined by the World Health Organization as one of the top ten deadly diseases in the world. Because of its hidden early symptoms, high degree of malignancy, and rapid deterioration, there is currently no effective way to completely treat tumors. In recent years, more and more studies have shown that the abnormality of cell cycle regulation-related genes is closely related to many diseases and malignant tumors. The prevention and treatment of malignant tumors are of great significance. [0003] The cell cycle is a highly ordered operation process, which is realized by regulating the cell cycle through the cell cycle-dependent kinase complex (Cyclin / CDKs). The formation of Cyclin / CDKs complex can promote cell proliferation, and the regulatory pathway of this complex is v...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12N15/12A61K45/00A61P35/00
CPCA61K45/00C07K14/4738C12Q1/6886C12Q2600/136C12Q2600/158
Inventor 代方银李念念童晓玲孟刚鲁成胡海韩民锦向仲怀
Owner SOUTHWEST UNIV
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