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Babesia orientilas thrombin gene 1 and protein coded by same

A technology of thrombin and babesia, applied in the field of molecular biology, can solve the problem of no diagnostic kit for oriental babesia and achieve good reactogenicity

Inactive Publication Date: 2016-04-27
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

To date, there are no diagnostic kits for the detection of babesiosis orientalis

Method used

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  • Babesia orientilas thrombin gene 1 and protein coded by same
  • Babesia orientilas thrombin gene 1 and protein coded by same
  • Babesia orientilas thrombin gene 1 and protein coded by same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1: the purification of Babesia orientalis

[0023] Put freshly infected red blood cells (infection rate is about 5-10%) into a sterile RNase-free centrifuge tube, centrifuge at low speed (3000rpm) for 5 minutes, collect blood cells and remove white flocs between supernatant and blood cells (white blood cells); add 3 times the volume of blood cells in 1xPBS, shake well, centrifuge at 3000rpm for 5 minutes, collect blood cells, repeat this operation once until the supernatant is colorless and transparent; add 2 times the volume of red blood cell lysate, shake well, and let stand at room temperature for 10 minutes , centrifuge at 10000rpm for 7min, collect the precipitate; add 3 times the volume of 1xPBS, shake well, centrifuge at 10000rpm for 7min, collect the precipitate, repeat this step until the supernatant is colorless and transparent; wash the precipitate once with sterile RNase-free PBS, add 1 / Dissolve 10 volumes of sterile RNase-free PBS, which is the ...

Embodiment 2

[0024] Embodiment 2: the extraction of total RNA of Babesia orientalis

[0025] All utensils for RNA extraction were treated with 0.1% DEPC water and baked at 150°C for 4 hours; plastic utensils were soaked in 0.1% DEPC water overnight and sterilized at 121°C for 20 minutes; metal utensils were soaked in 1mol / L NaOH for 2 hours, After thoroughly rinsing with 0.01% DEPC water, dry at 37°C; each reagent was prepared with 0.01% DEPC water without ribonuclease (RNase). Take 150 μL of Babesia orientalis purified by the above method to extract RNA. Total RNA was extracted according to the instructions of Trizol reagent. Extracted total RNA samples were frozen at -80°C for future use.

Embodiment 3

[0026] Embodiment 3: the synthesis of total cDNA of Babesia orientalis

[0027] Using TaKaRa's The ReverseTranscriptase Reverse Transcription Kit uses the extracted total RNA of Babesia orientalis as a template for reverse transcription. The specific steps are as follows:

[0028] ① Prepare the following mixture in Microtube

[0029]

[0030] ② Perform denaturation and annealing reactions under the following conditions on a PCR instrument:

[0031] 65℃5min↓Quick cooling on ice

[0032] ③ Prepare the following reverse transcription reaction solution in the above-mentioned Microtube tube.

[0033]

[0034] ④ Perform the reverse transcription reaction on the PCR instrument according to the following conditions:

[0035] 30℃10min

[0036] ↓

[0037] 42℃30-60min

[0038] ↓

[0039] After treatment at 95°C for 5 minutes (enzyme inactivation), place on ice.

[0040] The cDNA obtained by reverse transcription was stored at -20°C for future use.

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PUM

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Abstract

The invention discloses a babesia orientilas thrombin gene 1 which has the nucleotide sequence shown in SEQ ID NO:1. The invention further discloses recombinant protein obtained by coding the babesia orientilas thrombin gene 1, wherein the recombinant protein has the amino acid sequence shown in SEQ ID NO:2. The recombinant protein can effectively detect the existence of antigens of a water buffalo infected with the babesia orientilas, has good reactogenicity, belongs to a candidate factor for developing a serological diagnosis method for detecting the babesia orientilas disease, and is beneficial for controlling the prevalence and spread of the babesiosis disease of the water buffalo.

Description

technical field [0001] The invention belongs to the field of molecular biology and relates to a new gene and its recombinant protein, specifically Babesia orientalis thrombin gene 1 and its encoded recombinant protein. The invention also relates to the application of the recombinant protein. Background technique [0002] Buffalo babesiosis (Babesiosis) is a tick-borne blood protozoan disease caused by Babesia orientalis parasitizing on the erythrocytes of buffalo and characterized by high fever, anemia, jaundice and hemoglobinuria. The disease is widely prevalent in Hubei, Fujian, Jiangxi, Anhui, Jiangsu, Zhejiang, Hunan, Guangxi, Yunnan, Guizhou and other provinces in the south of my country, and has seriously affected the buffalo breeding industry and agricultural production. Since Babesia orientalis was first reported in 1984, its transmission and prevalence have shown a tendency to spread. There are three main reasons for the analysis: first, once an animal is infected w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/57C12N9/74C12Q1/37
CPCC12N9/64C12Q1/37C12Y304/21005
Inventor 贺兰赵俊龙喻龙何军伟何沛黄源
Owner HUAZHONG AGRI UNIV
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