Tick cystatin Rhcyst-2, and gene and applications thereof

A cysteine ​​protease, rhcyst-2 technology, applied in the field of bioengineering, can solve the problem that there is no research report on cysteine ​​protease inhibitory molecules of R. falciparum

Inactive Publication Date: 2014-08-06
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no research report on cys

Method used

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  • Tick cystatin Rhcyst-2, and gene and applications thereof
  • Tick cystatin Rhcyst-2, and gene and applications thereof
  • Tick cystatin Rhcyst-2, and gene and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Gene cloning and sequence analysis of RHcyst-2, a cysteine ​​protease inhibitor of Rhizocephalus falciparum

[0038] 1. Extraction of total RNA from female Rhizocephalus sylvestris half-saturated

[0039] Rhizocephalus falciparum was inoculated on rabbit ears at a ratio of 2:1 male to female, and half-full blooded female ticks were removed from rabbit ears 4 days later. Grind with liquid nitrogen on a sterilized mortar, then add 1ml of Trizol reagent to grind thoroughly and transfer to a nuclease-free EP tube. The total RNA of 4 Rhizocephalus falciparum half-saturated females was extracted by the classic Trizol method, the concentration was 11.3ug / ul, and the OD260 / OD280 value was 1.942, which could meet the requirements of subsequent experiments in terms of concentration and purity.

[0040] 2. Rapid amplification of the 3' end

[0041] 2.1 Primer design

[0042] For the family 2 cystatin molecule of Rhizocephalus falciparum, based on the conserved QVVAGXN...

Embodiment 2

[0086] Example 2 Prokaryotic expression and purification of the cysteine ​​protease inhibitor RHcyst-2 of Rhizocephalus falciparum

[0087] 1. Expression plasmid construction

[0088] According to the analysis results of the full-length sequence of RHcyst-2, gene-specific primers with appropriate enzyme cutting sites were designed at both ends of the ORF of the RHcyst-2 gene to remove the signal peptide. The primer sequences are as follows:

[0089] RHcyst-2express F: 5'-TA GAATTC CAACCGCTGGTTGGCGG-3' (SEQ ID NO. 9);

[0090] RHcyst-2express R:5'-TA CTCGAG CTAGGTGGATGCGCTGCT-3' (SEQ ID NO. 10).

[0091] PCR amplified the coding region of the RHcyst-2 gene to remove the signal peptide, cloned it into the pGEX-4T-1 vector, constructed and expressed the recombinant plasmid RHcyst-2 / pGEX-4T-1, and subjected to double digestion (Xho I and EcoR I) and sequencing Identify the accurate insertion of the target fragment. Figure 5 It is the double enzyme digestion identification...

Embodiment 3

[0109] Example 3 Enzyme Activity Analysis Experiment of Recombinant Protein rRHcyst-2

[0110] 1. Method: The activity of rRHcyst-2 on papain-like cysteine ​​protease was verified by measuring the residual activity of cathepsin L, B, C, H, S, papain and its corresponding fluorescent substrate after the action of rRHcyst-2. inhibitory activity. The specific method is as follows:

[0111] (1) The cysteine ​​protease reaction solution was prepared according to the formula of 100mM NaAC, 100mM NaCl, 1mM EDTA, 1mg / ml cysteine ​​and 0.005% TritonX-100, and the pH was adjusted to 5.5.

[0112] (2) Six cysteine ​​proteases were prepared to 1.5uM with cysteine ​​protease reaction solution, and their corresponding fluorescent substrates were prepared to 0.5mM.

[0113] (3) The purified RHcyst-2 recombinant protein was prepared into a concentration gradient of 12uM, 6uM, 3uM, 1.5uM, 0.75uM, 0.375uM and 0uM with PBS.

[0114] (4) Add 20ul of RHcyst-2 recombinant protein with seven conc...

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Abstract

The invention discloses a tick cystatin. The tick cystatin is a Rhipicephalus haemaphysaloide cystatin Rhcyst-2. The invention also discloses a Rhipicephalus haemaphysaloide cystatin Rhcyst-2 gene. The Rhipicephalus haemaphysaloide cystatin Rhcyst-1 gene includes a nucleotide sequence coding an amino acid sequence represented by SEQ ID NO.1. The Rhipicephalus haemaphysaloide cystatin Rhcyst-2 has substantial cysteine protease inhibition activity and toxoplasma infection resistance, and is suitable for preparing anti-parasitical medicines.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a tick cysteine ​​protease inhibitory molecule and its gene and application. Background technique [0002] Cysteine ​​proteases have important functions in the physiological metabolism of parasites and are considered to be a promising target for antiparasitic drugs. Chemical cysteine ​​protease inhibitors have been used to treat trypanosomes and malaria parasites. There are also inhibitory molecules with similar functions to chemical inhibitors in organisms, which are reversibly tightly bound to cysteine ​​proteases and efficiently inhibit enzyme activity, which are called cystatins. Studies have found that cysteine ​​protease inhibitory molecules in ticks are very rich, and because of their generally good inhibitory activity on cysteine ​​proteases, they may have the value of being developed as antiparasitic drugs. In addition, studies have found that the human cystatin...

Claims

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Application Information

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IPC IPC(8): C07K14/81C12N15/15C12N15/63C12N1/21A61K38/57A61P33/00A61P33/02A61P33/06
CPCA61K38/00C07K14/8139Y02A50/30
Inventor 周金林王玉俭周勇志曹杰张厚双龚海燕
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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