Construction of recin A chain mutant and application of recin A chain mutant as candidate vaccine antigen

A technology of vaccines and recombinant vectors, applied in antiviral agents, recombinant DNA technology, medical preparations containing active ingredients, etc.

Active Publication Date: 2011-06-01
MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Chinese scholars have just started to study the detection and medical protection of ricin biological warfare agents. There is no supporting diagnostic reagents and effective imm

Method used

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  • Construction of recin A chain mutant and application of recin A chain mutant as candidate vaccine antigen
  • Construction of recin A chain mutant and application of recin A chain mutant as candidate vaccine antigen
  • Construction of recin A chain mutant and application of recin A chain mutant as candidate vaccine antigen

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Experimental program
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Effect test

Embodiment 1

[0030] Embodiment 1, the acquisition of mutant mRTA

[0031] 1. Obtaining of mutant mRTA

[0032] According to the A chain (rRTA) gene sequence of existing ricin, its nucleotide sequence is the sequence 5 in the sequence listing, and the aminoacid sequence is the sequence 6 in the sequence listing, adopts Quickchange Lighting Site-Directed Mutagenesis Kit (Stratagene company , Catalog#210518) to obtain mutant mRTA-D75AV76MY80A (plasmid) by site-directed mutation, after sequencing, the gene contained in mRTA-D75AV76MY80A was named mRTA, its nucleotide sequence was sequence 1 in the sequence listing, and the coding region was sequence 1 in the sequence listing The 1st-801st nucleotide sequence from the 5' end of sequence 1, the amino acid sequence of mRTA protein encoded by mRTA is the sequence shown in sequence 2 in the sequence list. Using Lasergene software to predict the antigenicity of the constructed mutants did not change significantly.

[0033] The mutation results wer...

Embodiment 2

[0047] Embodiment 2, the biological activity detection of mutant mRTA

[0048] 1. Quantification of Mutant Proteins

[0049] The purified mRTA obtained in Example 1 was quantified using the BCA method protein quantification kit (purchased from Beijing Biotech Biotechnology Co., Ltd., Lot #20120613). The experiment was repeated three times, and the results were averaged.

[0050] Specific steps:

[0051] Kit composition: protein standard (5mg / mlBSA), Solution A, Solution B.

[0052] (1) When using, shake and mix Solution A. According to the number of samples, prepare an appropriate amount of BCA working solution with 50 volumes of Solution A plus 1 volume of Solution B (50:1), and mix well.

[0053] (2) Completely dissolve the protein standard (5mg / mlBSA), dilute it to 1.5mg / ml, 1.0mg / ml, 0.8mg / ml, 0.4mg / ml, 0.2mg / ml with PBS as the standard.

[0054] (3) Add the standard substance and the sample to be tested at each concentration mentioned above to the 96-well plate respect...

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Abstract

The invention discloses recin vaccine related protein, coded genes thereof and application thereof. The protein provided by the invention is named as mRTA and is shown as the following 1) or 2): 1) the protein consists of an amino acid residue sequence of a sequence 2 in a sequence table; or 2) the protein is derived from 1), has the same function, and is formed by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid residue sequence of the sequence 2 in the sequence table. A test proves that the mutant mRTA obtained by point mutation is the recin A chain mutant protein obtained by recombinant expression. According to detection, the recin A chain mutant protein has the characteristics of low toxicity, no vascular leakage, high immunogenicity and thelike and can serve as candidate vaccine antigen.

Description

technical field [0001] The invention relates to the construction of a ricin A chain mutant and its use as a candidate vaccine antigen, in particular to a ricin vaccine-related protein, its coding gene and its application. Background technique [0002] With the rapid development of biotechnology and the development and utilization of biotoxins, the importance of toxins in weapons of mass destruction has been highlighted, and the threat of biotoxins and their bioterrorism is increasing day by day. Ricin is composed of A and B chains, A chain is the effector chain, which has RNA N-glycosidase activity, and B chain is the binding chain, which mediates the A chain to enter the cell to play a toxic role. Ricin mice intraperitoneal LD 50 It is 3.0μg / kg, and its toxicity is at least 380 times that of organophosphate nerve agents. my country is one of the main production areas of castor in the world. The raw materials of castor bean have a wide range of sources and are easy to obta...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/63A61K38/16A61K48/00A61P39/02
Inventor 王景林韩艳辉高姗康琳
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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