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Ruditapes philippin RpTNF gene recombinant protein, its preparation and application

A Philippine clam and gene recombination technology, applied in the field of genetic engineering, can solve problems such as lack of TNF information, and achieve the effects of obvious activity characteristics, wide application range and simple operation

Active Publication Date: 2012-03-14
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, compared with the state of extensive and in-depth research on higher animals, the research on TNF in invertebrates has just started. Currently, apart from the gene sequences in individual species, other information on TNF is seriously lacking, especially the lack of a clear understanding of its function.

Method used

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  • Ruditapes philippin RpTNF gene recombinant protein, its preparation and application
  • Ruditapes philippin RpTNF gene recombinant protein, its preparation and application
  • Ruditapes philippin RpTNF gene recombinant protein, its preparation and application

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0020] The RpTNF gene recombinant protein of clam philippines is shown in the amino acid sequence of SEQ ID NO.1 in the sequence table.

[0021] SEQ ID NO.1

[0022] MDKKLKNISMFVLLLVVVCLMAVLALVWSMPDIPKTVKTSELCLPSEIGLLKCGSTVDLLHDYL

[0023] EREIATKYDEVKRQNQKKQENHLKEVSARLMNIYDKSFWEMKPAAKLTGKEQPDLRKTDSNADM

[0024] VPIREWRNGRELYDTKGFIRYGIRYRNGRLVVPVDGTYFIYSNIDFFESCNPSSGLPDIKDINK

[0025] PIKHGIFKFNILEGEENELVSNVQPHTISTNRYYNSYSSYVSSLAELKAGDELSVKVSNITYIR

[0026] YTRDNYFGVNLI

[0027] (a) Sequence features:

[0028] ●Length: 268 amino acids

[0029] ●Type: protein

[0030] ●Chain type: double chain

[0031] (b) Molecular type: double-stranded DNA

[0032] (c) Assumption: No

[0033] (d) Antisense: no

[0034] (e) Original source: Philippine clams

[0035] (f) Specific name: PRT

[0036] Construction of RpTNF gene recombinant protein of clam philippines:

[0037] 1. Construction of the recombinant vector: The recombinant vector used in the present invention is the pET21a...

example 2

[0050] Anti-tumor Growth Activity Analysis of Recombinant Proteins

[0051] Taking tumor cell 7402 as the target cell, the killing effect of the RpTNF protein obtained in Example 1 on tumor cells was detected. Adjust tumor cell concentration to 1-5 x 10 5 / ml, take a 96-well plate and add 100ul cell suspension to each well, place at 37°C, 5% CO 2 Incubate overnight in an incubator under saturated humidity conditions; on the second day, different concentrations of recombinant proteins were added to the cell culture medium in advance, and 6 replicate wells were set up in each group, and a cell control group (without any addition) and a blank well (only added) were set up. culture medium); put the culture plate into a CO2 incubator, and cultivate for 72 hours at 37°C, 5% CO2 and saturated humidity; add 10ul of MTT solution (5mg / ml) to each well, and continue to cultivate for 4h at 37°C, protected from light; Terminate the culture, carefully discard the culture supernatant in th...

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Abstract

The invention, relating to genetic engineering, particularly relates to Ruditapes philippin RpTNF gene recombinant protein, its preparation and application. The RpTNF gene recombinant protein is shown as an amino acid sequence in SEQ ID No.1 in the sequence table. The preparation is characterized by amplifying Ruditapes philippin tumor necrosis factor RpTNF gene cDNA full length through PCR technique, and carrying out prokaryotic recombination expression on the TNF structure. According to the invention, the preparation has simple operation, the constructing process of the vector can be completed with need of routine connection, conversion and sequencing and screening; the obtained recombinant protein has obvious activity characteristics, e.g. the RpTNF protein disclosed herein is the first sea invertebrate cytokine having biological activity.

Description

technical field [0001] The invention relates to genetic engineering, in particular to a philippine clam RpTNF gene recombinant protein and its preparation and application. Background technique [0002] Cytokine is a general term for biologically active small molecule proteins secreted by immunocompetent cells and other cells, and is the most diverse and important class of immune effector molecules involved in the innate immune response. At present, hundreds of cytokines have been discovered, and a lot of research has been done on their source, molecular structure, corresponding receptors, signal transduction, biological function and clinical relationship, especially the recombinant cytokines produced by genetic engineering technology have been It has been successfully applied to the treatment of diseases such as tumors, inflammation, and hematopoietic dysfunction, and has very broad application prospects. [0003] In view of the extensive immunomodulatory effects of cytokin...

Claims

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Application Information

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IPC IPC(8): C07K14/525C12N15/28C12N15/70A61K38/19A61P35/00
Inventor 邱丽梅宋林生王玲玲赵建民
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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