B.canis BcMSA1-BcSA1 recombinant protein and preparation method and application thereof

A technology of Babesia canis, bcmsa1-bcsa1, which is applied in the field of Babesia canis BcMSA1-BcSA1 recombinant protein and its preparation, and achieves the effects of wide application prospect, no training and improved sensitivity

Pending Publication Date: 2021-03-05
杭州爱谨生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, current assays and tools for the detection of babesiosis infection by serological tests still have major limitations

Method used

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  • B.canis BcMSA1-BcSA1 recombinant protein and preparation method and application thereof
  • B.canis BcMSA1-BcSA1 recombinant protein and preparation method and application thereof
  • B.canis BcMSA1-BcSA1 recombinant protein and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 Construction of Babesia canis BcMSA1-BcSA1 fusion protein gene expression vector

[0063] The BcMSA1 gene of Babesia canis was designed according to the protein sequence of NCBI Gene bank: KR134351. According to the hydrophilicity and hydrophobicity of the protein ( https: / / web.expasy.org / protscale / ) analysis, the BcMSA1 (1-70aa) sequence was selected for fusion in the area predicted to have a higher hydrophilic content. The BcSA1 gene was designed according to the protein sequence of NCBI Gene bank: KR134352. According to the hydrophilicity and hydrophobicity of the protein ( https: / / web.expasy.org / protscale / ) analysis, the BcSA1 (10-250aa) sequence was selected for fusion in the predicted region with higher hydrophilic content.

[0064] The amino acid sequence of the BcMSA1-BcSA1 recombinant protein fusion protein is as follows (SEQ ID NO.1):

[0065] MMLLFALSTLVTFAFCDGENTILLSNVEFHTPVSSVKLLKEYSSNQESMAVIMMLTEMPNTSGKLTDGKVHPHNFILIFQLLATMGNAQSTSSQENSRDG...

Embodiment 2

[0070] Example 2 Expression of Babesia canis BcMSA1-BcSA1 fusion protein

[0071] Transform the Babesia BcMSA1-BcSA1 fusion gene plasmid into Escherichia coli BL21, spread it on an LB plate containing 50 μg / mL kanamycin (Shanghai Sangong, product number: K0408), culture overnight at 37°C, and pick a single Cloned colonies were cultured at 37°C in 300 mL LB medium containing the same concentration of kanamycin until the OD600 reached about 0.6, and the expression was induced with IPTG (Shanghai Shengong, product number: IB0168) with a final concentration of 1 mM. The induction conditions were: 25°C, rotation speed 200rpm, 4h. After induction, the culture solution was centrifuged at 4° C. at 7000 rpm for 10 min to collect the bacteria.

Embodiment 3

[0072] Example 3 Purification and renaturation of Babesia canis BcMSA1-BcSA1 fusion protein

[0073] Use 50mL loading buffer Binding Buffer (50mM Tris, 0.2M Nacl, pH8.0) 50mL to crush the bacteria; then ultrasonically break, the condition is 600w, ultrasonic 2s, interval 5s, a total of 100 times; finally 12000rpm, 30min, 4℃ Collect the supernatant by centrifugation, and the target protein is in the supernatant. Then, it was purified by Ni column, and the target protein was eluted with Elution Buffer (50mM Tris, 0.2M Nacl, 0.5M Imidazole, pH8.0). The target protein was detected by PAGE gel electrophoresis, and the results were as follows figure 1 shown.

[0074] Depend on figure 1 It can be seen that the purified fusion protein has a high purity. The purified recombinant protein was dialyzed with a dialysis buffer (50mM Tris, 0.2M Nacl, pH8.0), and the dialysis solution was changed every 12 hours for a total of 3 times. The dialyzed protein solution was taken out, filtered ...

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Abstract

The invention discloses a B.canis BcMSA1-BcSA1 recombinant protein, and belongs to the field of animal virus antibody detection. The recombinant protein comprises an amino acid sequence shown in SEQ ID NO. 1 or is composed of the amino acid sequence shown in SEQ ID NO. 1. The invention further discloses a gene of the recombinant protein, a carrier and a host cell containing the gene, a preparationmethod of the recombinant protein and application of the recombinant protein to detection of an antibody of B.canis. The recombinant protein is used for detecting the antibody of B.canis, is convenient and fast to use, high in sensitivity, free of cross reaction with other pathogens and high in specificity, and has great clinical significance and wide application prospects.

Description

technical field [0001] The invention belongs to the field of animal virus antibody detection, and in particular relates to a Babesia canis BcMSA1-BcSA1 recombinant protein and its preparation method and application. Background technique [0002] Babesia (Babesia) belongs to the genus Babesia, which includes more than 90 kinds of Babesia. It has been reported that there are many types of Babesia that can infect dogs, mainly B. vogeli, Babesia Gibsoni (B.gibsoni), Babesia canis (B.canis), Babesia rossi (B.rossi) and Babesia double buds (B.bigemina), etc. Among them, the body size of Babesia canis is 3-5 μm, and it exists in single or paired form. [0003] Babesia can cause tissue and organ damage. It will damage the red blood cells of dogs. The urine of sick dogs will turn red. Symptoms such as high fever, anemia, and rapid weight loss will also appear. If not diagnosed and treated in time, the sick dog will have the risk of death . Generally speaking, the incubation period...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21G01N33/68G01N33/569G01N33/558G01N33/543C12R1/19
CPCC07K14/44C07K2319/00C12N15/70G01N33/54346G01N33/558G01N33/56905G01N33/68G01N2333/44
Inventor 李晓光
Owner 杭州爱谨生物科技有限公司
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