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Tick ​​autophagy-related protein molecule atg5 and its application

A technology of autophagy-related proteins and molecules is applied to the tick autophagy-related protein molecule ATG5 and its application fields, which can solve the problems of no reports and achieve the effect of broad application prospects.

Active Publication Date: 2022-07-15
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

Autophagy and apoptosis are considered to be the two main factors that induce the degeneration of tick salivary glands. In mammals and arthropod insects, the autophagy-related protein ATG5 has been confirmed to be the molecular switch from autophagy to apoptosis. This molecule has not been reported in ticks

Method used

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  • Tick ​​autophagy-related protein molecule atg5 and its application
  • Tick ​​autophagy-related protein molecule atg5 and its application
  • Tick ​​autophagy-related protein molecule atg5 and its application

Examples

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Effect test

Embodiment 1

[0024] Example 1 Gene cloning and sequence analysis of R. falciparum autophagy-related protein ATG5 (RhATG5)

[0025] 1. Materials and methods

[0026] 1.1. Ticks and laboratory animals

[0027] R. falciparum ticks were collected from Wuchang, Hubei Province, and were artificially reared and preserved by rabbits in this laboratory. 6-week-old male Kunming mice were purchased from Shanghai Slack Experimental Animal Co., Ltd. New Zealand white rabbits were purchased from the Experimental Animal Center of Fudan University.

[0028] 1.2. Bacteria and Plasmids

[0029] Escherichia coli DH5α and BL21(DE3) competent cells (Transgen) were used for plasmid construction and protein expression. The gene cloning vector is pMD-18T (Takara), and the expression vector is pET30a vector (Novagen) and pCMV-HA (Takara).

[0030] 1.3 Molecular cloning of Rh ATG5

[0031] TRIzol reagent (Invitrogen) was used to extract RNA from salivary glands of blood-saturated ticks after microdissection. ...

Embodiment 2

[0034] Example 2 Expression and purification of recombinant protein

[0035] In order to achieve a higher purification level, an expression vector is constructed so that the N-terminal of the target protein has a His tag.

[0036] According to the pET30a vector vector sequence and the Rh ATG5 full-length gene, two enzyme cleavage sites, BamHI and Hind III, and a fragment of part of the vector sequence were introduced by primer design. Subcloned into the double-enzyme digested expression vector pET-30a (Novagen), and sequenced to ensure the accuracy of the sequence. The ligation products were transformed into competent cells E. coli BL21 (DE3) for expression. The expressing bacteria were induced by 1 mM IPTG, incubated at 37°C for about 10 hours, and then the cells were collected and stored at -80°C. The recombinant protein was purified by Ni-NTA His·Bind Resin (Novagen), and the collected induced bacterial cell pellets were resuspended with bindingbuffer (NI-NTA Buffer Kit, ...

Embodiment 3

[0038] Example 3 Detection of native protein RhATG5 in tick salivary glands at different blood-sucking times

[0039] 1. Preparation of recombinant protein RhATG5 antiserum and detection by western blotting

[0040]The purified recombinant protein was mixed with an equal volume of Freund's complete adjuvant and emulsified, and mice were immunized by subcutaneous injection (about 100 μg of recombinant protein per mouse), and then boosted every two weeks for a total of two times, and the recombinant protein required Mix equal volume with incomplete adjuvant to emulsify. Blood samples from mice were collected by orbital blood sampling one week after the third immunization, and antibody titers were detected by ELISA.

[0041] Tick ​​salivary gland samples with different blood sucking time were quantified by brandford, adjusted to the same concentration, mixed with 2×SDS gel-loading buffer, and then denatured at 100°C for 10 minutes. After gel electrophoresis on 12% SDS-PAGE, it ...

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Abstract

The invention discloses a tick autophagy-related protein molecule ATG5, which has the amino acid sequence shown in SEQ ID NO.1. The invention also discloses the gene of the tick autophagy-related protein molecule ATG5, which comprises: a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.1. The tick autophagy-related protein molecule ATG5 and its gene of the present invention can significantly affect the blood-sucking of ticks after gene silencing, and are expected to be candidate molecules for the prevention and treatment of tick-borne diseases, and have broad application prospects.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a tick autophagy-related protein molecule ATG5 and its application. Background technique [0002] Ticks are obligate blood-sucking (blood-feeding) arthropods that are widely distributed around the world. Ticks can infect almost all terrestrial vertebrates, including mammals, birds, various reptiles and amphibians, and are vectors of many diseases. All ticks have 4 stages: egg and three active stages (juvenile, nymph and adult). Rhipicephalushaemaphysaloides (R. falciparum) is one of the tick species widely distributed in southern China. It belongs to the Ixodes family. It is a three-host tick, and the Ixodes tick needs to change hosts at each stage of its life cycle. The salivary glands of ticks play an important role in the blood-sucking process of ticks, and are the main mediators for the transmission of tick-borne pathogens. It usually takes 7 to 14 days for ticks to ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C12N15/12
CPCC07K14/43527
Inventor 周金林王亚楠龚海燕张厚双曹杰周勇志
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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