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Autophagy related protein molecule ATG5 of rhipicephalus haemaphysaloides and application thereof

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

Active Publication Date: 2020-02-14
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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  • Autophagy related protein molecule ATG5 of rhipicephalus haemaphysaloides and application thereof
  • Autophagy related protein molecule ATG5 of rhipicephalus haemaphysaloides and application thereof
  • Autophagy related protein molecule ATG5 of rhipicephalus haemaphysaloides and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

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

[0025] 1. Materials and methods

[0026] 1.1. Ticks and experimental animals

[0027] Rhizocephalus falciparum was collected from Wuchang, Hubei, and was preserved in our laboratory by artificial rearing of rabbits. Six-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), pCMV-HA (Takara).

[0030] 1.3 Molecular cloning of Rh ATG5

[0031] TRIzol reagent (Invitrogen) was used to extract salivary gland RNA from engorged ticks after microdissection. ...

Embodiment 2

[0034] Example 2 Expression and purification of recombinant protein

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

[0036] According to the vector sequence of pET30a vector and the full-length Rh ATG5 gene, two restriction sites, BamHI and Hind III, and a fragment of the vector sequence were introduced by primer design. The method refers to the In-Fusion HD Cloning Kits (Clontech, Takara Bio) kit instructions, Subcloned into the expression vector pET-30a (Novagen) after double digestion, and sequenced to ensure the accuracy of the sequence. The ligation product was transformed into competent cells E.coli BL21(DE3) for expression. The expression bacteria were induced by 1mM IPTG, and after incubation at 37°C for about 10 hours, 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 c...

Embodiment 3

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

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

[0040]The purified recombinant protein was mixed with an equal volume of Freund's complete adjuvant and then emulsified, then immunized mice by subcutaneous injection (about 100 μg of recombinant protein each), and then boosted once every two weeks, twice in total, and the recombinant protein required Mix and emulsify with equal volume of incomplete adjuvant. One week after the third immunization, mouse blood samples were collected by orbital blood collection, and antibody titers were detected by ELISA.

[0041] Tick ​​salivary gland samples with different blood-sucking times were quantified by Brandford and 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 an autophagy related protein molecule ATG5 of rhipicephalus haemaphysaloides. The autophagy related protein molecule ATG5 has an amino acid sequence as shown in SEQ ID NO.1. The invention further discloses a gene of the autophagy related protein molecule ATG5 of the rhipicephalus haemaphysaloides. The gene includes a nucleotide sequence encoding the amino acid sequence as shown in the SEQ ID NO.1. The autophagy related protein molecule ATG5 of the rhipicephalus haemaphysaloides and gene thereof can remarkably influence blood sucking of the rhipicephalus haemaphysaloidesafter gene silencing. The autophagy related protein molecule ATG5 is expected to become a candidate molecule for prevention and control of tick-borne diseases and has a broad application prospect.

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 widely distributed throughout 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 (larva, nymph and adult). Rhipicephalushaemaphysaloides is one of the tick species widely distributed in southern China. It belongs to the Ixodes family. The salivary glands of ticks play an important role in the blood-sucking process of ticks, and are the main organ for the transmission of tick-borne pathogens. It usually takes 7-14 days for ticks to become full of blood from the upper body. Male ticks can repeat intermittent blood-sucking, while female ticks cannot. The upper b...

Claims

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Application Information

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Patent Type & Authority Applications(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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