Globoid protein gene 4 of babesia orientalis and protein for encoding globoid protein gene 4

A technology for Babesia and Babesiosis, applied in the field of molecular biology, can solve the problems of complicated operation, easy misjudgment, difficult clinical detection, etc., and achieve the effect of good immunogenicity

Inactive Publication Date: 2018-09-28
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Among them, microscopy detection requires the detection personnel to have rich clinical experience, and when the sample to be tested has a low rate of insect infection or mixed infection, misjudgment is prone to occur; molecular biology detection methods have the characteristics of high sensitivity and strong specificity, but often require expensive Advanced instruments such as PCR instruments are complicated to operate and are difficult to use in clinical testing; while the serological method of Babesia orientalis is still in the stage of laboratory research and development, and is rarely used clinically

Method used

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  • Globoid protein gene 4 of babesia orientalis and protein for encoding globoid protein gene 4
  • Globoid protein gene 4 of babesia orientalis and protein for encoding globoid protein gene 4
  • Globoid protein gene 4 of babesia orientalis and protein for encoding globoid protein gene 4

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1: the purification of Babesia orientalis

[0024] Collect 100mL anticoagulant blood from buffalo infected with insects (infection rate is about 5-10%) in two 50mL centrifuge tubes, centrifuge at 3000rpm at low speed for 10min, remove the white flocs (leukocytes) between the supernatant and red blood cells; slowly add 3 times the volume of red blood cells in 1xPBS, shake well, repeat this operation 3 times until the supernatant is colorless and transparent; add 2 times the volume of red blood cell lysate, shake well, let stand at room temperature for 30 minutes, blow and beat the 10mL syringe repeatedly 10 times, centrifuge at 12000rpm for 10 minutes, Collect the precipitate and repeat this step 3 times until the supernatant is colorless and transparent; the precipitate is dissolved in 1xPBS, which is Babesia orientalis.

Embodiment 2

[0025] Embodiment 2: the extraction of Babesia orientalis gDNA

[0026] The extracted parasite solution was used for the extraction of gDNA, and the extraction kit was Tiangen kit. The extracted gDNA samples were frozen at -20°C for future use.

Embodiment 3

[0027] Example 3: Cloning and sequence analysis of Babesia orientalis globular protein gene 4

[0028] 1) Primer design

[0029] The upstream and downstream primers F1: 5'-ATGGTGGCCTCTTTCCCTACG-3'; R1: 5'-TTACTCAGTGGTGGTTTCGGTTTC-3' were designed using clone manager software.

[0030] 2) PCR amplification: use Babesia orientalis gDNA as a template, and perform PCR amplification with F1 and R1 primers. The reaction system is 25 μL. The PCR reaction system is as follows:

[0031]

[0032]

[0033] PCR reaction conditions: the reaction conditions are 94°C for 5min; 94°C for 30sec, 55°C for 90sec, 68°C for 1min; 72°C for 10min; 35 cycles.

[0034] 3) PCR product identification: After the amplification is completed, take 10 μL of the PCR product and apply it with 10×nucleic acid loading buffer, 1.0% agarose gel, 1×TAE buffer, 120V, electrophoresis for 30 minutes to observe the results, and obtain the target fragment (See figure 1 ).

[0035] 4) Cloning, screening and sequ...

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Abstract

The invention discloses a globoid protein gene 4 of babesia orientalis. The gene has a nucleotide sequence as shown in SEQ ID NO: 1. The invention further discloses a protein for encoding the globoidprotein gene 4 of the babesia orientalis. The protein has an amino acid sequence as shown in SEQ ID NO: 2. The protein can effectively detect the existence of a babesia orientalis antigen in a buffalobody, has high immunogenicity, can detect the existence of the babesia orientalis by applying an indirect immunofluorescence method, and can be used as a candidate molecule for diagnosing an antigen.

Description

technical field [0001] The invention belongs to the field of molecular biology and relates to a new Babesia orientalis gene and its application. Background technique [0002] Babesia orientalis is a parasitic protozoan that is transmitted by Rhizocephalus falciparum and only causes babesiosis in buffaloes. The insect was first discovered in central and southern China in 1984, and was officially named Babesia orientalis in 1997. The buffalo babesiosis caused by it mainly occurs in many regions in the south, and the onset season is mostly in May and June. It is related to the activity of Rhizocephalus falciparum, and has significant regional and seasonal characteristics. The clinical symptoms of the diseased buffalo are mainly fever, anemia, jaundice and hemoglobinuria, and even death in severe cases. If the diagnosis and treatment are not timely, the mortality rate will be higher, causing huge economic losses in the buffalo farming industry. At present, the diagnostic meth...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C07K14/44G01N33/68G01N33/569
CPCC07K14/44G01N33/56905G01N33/68G01N2333/44
Inventor 贺兰赵俊龙郭佳莹敖阳思琦王森
Owner HUAZHONG AGRI UNIV
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