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Anaplasma phagocytophilum P44 recombinant protein and preparation method and application thereof

A recombinant protein and phagocytic cell technology, applied in the fields of biochemical equipment and methods, virus/phage, recombinant DNA technology, etc., can solve the problems of missed detection and difficult grass-roots promotion, and achieve no need for expensive instruments, no pollution training, and improved sensitivity. Effect

Pending Publication Date: 2021-03-23
杭州亿米诺生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above methods such as immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR) technology require the use of designated equipment, corresponding test conditions and skills, and are difficult to promote at the grassroots level
[0004] In terms of the detection of Anaplasma phagocytophilum antibodies, the main research direction at home and abroad is to use a small part of the protein sequence to detect antibodies, which is bound to have the risk of missed detection

Method used

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  • Anaplasma phagocytophilum P44 recombinant protein and preparation method and application thereof
  • Anaplasma phagocytophilum P44 recombinant protein and preparation method and application thereof
  • Anaplasma phagocytophilum P44 recombinant protein and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 Construction of Canine Phagocytophilum Anaplasma P44 Fusion Protein Gene Expression Vector

[0062] The P44 gene of Anaplasma canis phagocytophilum was designed according to the protein sequence of NCBI Gene bank: WP_011451309.

[0063] The amino acid sequence of the P44 recombinant protein fusion protein is as follows (SEQ ID NO.1):

[0064] MMSLAIVMAGNDVRAHDDVSALDTGGAGYFYVGLDYSPAFSKIRDFSIRESNGETKAVYPYLKDGKSVKLESHKFDWNTPDPRIGFKDNMLVAMEGSVGYGIGGARVELEIGYERFKTKGIRDSGSKEDEADTVYLLAKELAYDVVTGQTDKLTAALAKTSGKDIVQFANAVKITNSTIDGKVCSGKHAALVPNKGKDYDADAKESNTNAHKTAQCSGLADSAATGPKSFSGFVGAVKVGEGKNWPTGRAASATSNETVVGPTNSNATAVAKDLVALNSDEKTIVAGLLAKTIEGGEVVEIRAVSSTSVMVNACYDLLSEGLGVVPYACVGLGGNFVGVVDGHITPKLAYRLKAGLSYQLSPEISAFAGGFYHRVVGDGVYDDLPAQRLVDDTSPAGRTKDTAIANFSMAYVGGEFGVRFAF

[0065] Since different species use different frequencies of synonymous codons, this codon bias has an impact on the translation process. If an mRNA has many clusters of rare codons, this can negatively a...

Embodiment 2

[0069] Example 2 Expression of Anaplasma P44 fusion protein in canine phagophilic cells

[0070] Transform the Anaplasma canis phagocytophilum P44 fusion gene plasmid into Escherichia coli BL21, smear it on an LB plate containing 50 μg / mL kanamycin (Shanghai Sangong, product number: K0408), culture overnight at 37°C, pick Monoclonal 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 Shenggong, 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

[0071] Example 3 Purification and renaturation of canine phagophilus Anaplasma P44 fusion protein

[0072] Use 50mL of loading buffer Binding Buffer (50mM Tris, 0.2M Nacl, pH8.0) 50mL to break up the bacteria; then ultrasonically break, the condition is 550w, ultrasonic 2s, interval 5s, a total of 100 times; finally 12000rpm, 30min, 4°C 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.

[0073] 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 o...

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Abstract

The invention discloses an anaplasma phagocytophilum P44 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 vector and a host cell containing the gene, a preparation method of the recombinant protein and application of the recombinant protein in detection of an anaplasma phagocytophilum antibody. The recombinant protein is used for detecting the anaplasma phagocytophilum antibody, is convenient and fast to use, high in sensitivity and high in specificity, has no cross reaction with other pathogens, 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 canine phagocyte Anaplasma P44 recombinant protein and a preparation method and application thereof. Background technique [0002] Canine Anaplasma phagocytophilum (Anaplasma phagocytophilum) is a blood pathogen caused by ticks as a transmission medium. The disease caused by it is a zoonotic disease transmitted by ticks. It is caused by neutrophils in the peripheral blood of animals. The clinical features of the disease are fever, general malaise, loss of appetite, fatigue and other flu-like symptoms. [0003] At present, the more direct diagnostic methods in clinical practice are blood smear staining, indirect fluorescence antibody test (Indirectfluorescence antibody, IFA) and enzyme-linked immunosorbent assay (Enzyme-Linked immunosorbentassay, ELISA) and other immunological methods, in addition to PCR (polymerase chain The method is also commonly us...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21C12P21/02G01N33/68G01N33/569G01N33/558C12R1/19
CPCC07K14/29C12N15/70G01N33/6854G01N33/56911G01N33/558C07K2319/21C12N2800/22G01N2333/29G01N2469/20
Inventor 李晓光何坚锋王哲侃
Owner 杭州亿米诺生物科技有限公司
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