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Recombinant chromatin modified albumin 1A, as well as coding gene and application thereof

A chromatin modification and protein technology, applied in the field of recombinant proteins, can solve the problems of poor anti-tumor effect, difficult to import into target cells, and difficult separation and purification of Chmp1A protein, achieving simple operation, high protein purity, high stability and activity. Effect

Active Publication Date: 2012-10-10
ZHEJIANG ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0016] The invention provides a recombinant chromatin modification protein 1A, which solves the problem that Chmp1A protein is difficult to separate and purify and is difficult to import into target cells, resulting in poor anti-tumor effect

Method used

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  • Recombinant chromatin modified albumin 1A, as well as coding gene and application thereof
  • Recombinant chromatin modified albumin 1A, as well as coding gene and application thereof
  • Recombinant chromatin modified albumin 1A, as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Extraction of Human Placenta Tissue mRNA

[0052] Take 50ug of human placental tissue preserved in liquid nitrogen, use the Trizol kit to extract the total RNA of human placental tissue in one step, and treat it with DNase (RNase-free) to remove the residual plasmid DNA in the muscle.

Embodiment 2

[0053] Example 2 RT-PCR amplification of human Chmp1A

[0054] Take 5u1 human placental tissue total RNA, use primer oligo(dT) for reverse transcription, and synthesize the first-strand cDNA; use cDNA as template, use primers P3 and P4 for PCR amplification, and finally the PCR product is passed through 1% agarose gel Electrophoretic analysis.

[0055] P3: 5'-CGGAATTCGCCATGGACGATACCCTGTT-3'

[0056] P4: 5'-TTGTCGACCGGGGCACGGC TAGTTCCTCAA-3'

[0057] PCR reaction system: 5μl of 10×PCR Buffer, 2μl of 10mM dNTPs, 2μl of P3 and P4 primers, 1μl of cDNA, 2μl of Taq plus DNA polymerase, 36μl of ddH2O, a total of 50μl.

[0058] PCR reaction conditions: 30 cycles of 94°C for 45s, 54°C for 45s, 72°C for 1min, and finally 72°C for 10min.

Embodiment 3

[0059] Example 3 Construction and sequence determination of sequencing vector

[0060] Purify the above RT-PCR product with a PCR product purification kit, connect it directly to the pGEM-T Easy vector using the T-A cloning strategy, transform the ligated product into Top10 competent cells, and screen on LB plates containing ampicillin, X-gal and IPTG . The positive clones identified by PCR and enzyme digestion were sequenced and identified. The results of Chmp1A sequencing and the deduced amino acid sequence are shown in Figure 8 .

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PUM

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Abstract

The invention discloses a recombinant chromatin modified albumin 1A, as well as a coding gene of and an application of the recombinant chromatin modified albumin 1A, wherein the recombinant chromatin modified albumin 1A comprises chromatin modified albumin 1A, the C end of the chromatin modified albumin 1A is connected with His-Tag, the N end of the chromatin modified albumin 1A is connected with a PTD (protein transduction domain) sequence, the amino acid sequence of the PTD sequence is YGRKKRRQRRR, and the amino acid sequence of the recombinant chromatin modified albumin 1A is shown as SEQ ID NO.2. According to the recombinant chromatin modified albumin 1A, an HIS-Tag tag is guided into wild type Chmp1A, so that the structure of the Chmp1A can not be changed, and can be easily purified through a Ni-NTA (nitrilotriacetic acid) Argarose albumen purifying system. The PTD sequence is guided into the wide type Chmp1A, so that the Chmp1A can effectively enter into tumor cell under the guide of the PTD, and the antitumor activity of the Chmp1A can be played.

Description

technical field [0001] The present invention relates to a recombinant protein, in particular to a recombinant chromatin modifying protein 1A and its coding gene and application. Background technique [0002] (1) Biological characteristics of Chmp1A [0003] Chromatin modifying protein 1A (Chromatin modifying protein 1A, Chmp1A) is a member of the ESCRT-III complex family. ESCRT-III components and their related proteins are collectively called Chmps. All Chmps proteins have a similar structure: they all encode an ORF of about 200 amino acids; they have a coiled-coil region and ujnnnnnn charge groups, and the basicity is located at the N-terminus. The acidity is at the C-terminus. Chmp1A is highly conserved in mammals, with a full-length open reading frame (ORF) of 591 nucleotides, encoding 197 amino acids. Chmp1A plays a key role in the formation of vesicles and multivesicular bodies (MVBs) and is directly associated with early and late endosomal membrane structure. [00...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C12N15/12C12N15/81C12N1/19A61K38/17A61K47/48A61P35/00C12R1/84
Inventor 由振强宣尧仙辛艳飞张立将陈云祥陈国灿杨红忠陈颖周国亮李峰徐潘生
Owner ZHEJIANG ACAD OF MEDICAL SCI
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