Tick cystatin Rhcyst-1, and gene and applications thereof

Abstract

The invention discloses a tick cystatin. The tick cystatin is a Rhipicephalus haemaphysaloide cystatin Rhcyst-1. The invention also discloses a Rhipicephalus haemaphysaloide cystatin Rhcyst-1 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-1 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...

Examples

Embodiment 3

[0124] Example 3 Enzyme Activity Analysis Experiment of Recombinant Protein rRHcyst-1

[0125] 1. Method: To verify the effect of rRHcyst-1 on papain-like cysteine ​​protease by measuring the remaining activity of cathepsin L, B, C, H, S, papain and its corresponding fluorescent substrate after the action of rRHcyst-1 inhibitory activity. The specific method is as follows:

[0126] (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.

[0127] (2) Six cysteine ​​proteases were prepared to 1.5uM with cysteine ​​protease reaction solution, and their corresponding fluorescent substrates were prepared to 0.5mM.

[0128] (3) The purified RHcyst-1 recombinant protein was formulated into a concentration gradient of 12uM, 6uM, 3uM, 1.5uM, 0.75uM, 0.375uM and 0uM with PBS.

[0129] (4) Add 20ul of RHcyst-1 recombinant protein with seven concent...

Embodiment 4

[0133] Example 4 Anti-Toxoplasma gondii infection experiment of recombinant protein rRHcyst-1

[0134] (1) The experimental animals are Kunming mice, about 25 grams in weight, male. Toxoplasma gondii is the international standard strain RH strain, ascites fluid was collected after infection of mice to collect parasites.

[0135] (2) RHcyst-1 recombinant protein was dissolved in PBS buffer, and the test was divided into high-dose group (200ug / only), medium-dose group (100ug / only), low-dose group (50ug / only), protein control group (GST protein) and a blank control group (PBS), each group of 20 test mice, each mouse was inoculated with 10,000 Toxoplasma gondii intraperitoneally. The recombinant protein was treated by intraperitoneal injection on the 2nd and 4th day after infection with Toxoplasma gondii, and then the mortality and time of death of the mice were observed. The average death time between groups was analyzed by t test to evaluate the effect.

[0136] The mouse mode...

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...

Examples

Embodiment 3

[0124] Example 3 Enzyme Activity Analysis Experiment of Recombinant Protein rRHcyst-1

[0125] 1. Method: To verify the effect of rRHcyst-1 on papain-like cysteine ​​protease by measuring the remaining activity of cathepsin L, B, C, H, S, papain and its corresponding fluorescent substrate after the action of rRHcyst-1 inhibitory activity. The specific method is as follows:

[0126] (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.

[0127] (2) Six cysteine ​​proteases were prepared to 1.5uM with cysteine ​​protease reaction solution, and their corresponding fluorescent substrates were prepared to 0.5mM.

[0128] (3) The purified RHcyst-1 recombinant protein was formulated into a concentration gradient of 12uM, 6uM, 3uM, 1.5uM, 0.75uM, 0.375uM and 0uM with PBS.

[0129] (4) Add 20ul of RHcyst-1 recombinant protein with seven concent...

Embodiment 4

[0133] Example 4 Anti-Toxoplasma gondii infection experiment of recombinant protein rRHcyst-1

[0134] (1) The experimental animals are Kunming mice, about 25 grams in weight, male. Toxoplasma gondii is the international standard strain RH strain, ascites fluid was collected after infection of mice to collect parasites.

[0135] (2) RHcyst-1 recombinant protein was dissolved in PBS buffer, and the test was divided into high-dose group (200ug / only), medium-dose group (100ug / only), low-dose group (50ug / only), protein control group (GST protein) and a blank control group (PBS), each group of 20 test mice, each mouse was inoculated with 10,000 Toxoplasma gondii intraperitoneally. The recombinant protein was treated by intraperitoneal injection on the 2nd and 4th day after infection with Toxoplasma gondii, and then the mortality and time of death of the mice were observed. The average death time between groups was analyzed by t test to evaluate the effect.

[0136] The mouse mode...

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