Rabbit hemorrhagic disease virus VP10-VP60 recombinant protein as well as preparation method and application of rabbit hemorrhagic disease virus VP10-VP60 recombinant protein

A recombinant protein and host cell technology, applied in biochemical equipment and methods, viruses, viral peptides, etc., can solve the problems of difficult promotion, time-consuming and other problems, and achieve the effect of improving sensitivity, low cost and strong specificity

Pending Publication Date: 2021-03-12
杭州爱谨生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The above method not only takes a long time, but also requires the use of designated instruments and equipment, corresponding test conditions and skills, and it is difficult to promote at the grassroots level

Method used

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  • Rabbit hemorrhagic disease virus VP10-VP60 recombinant protein as well as preparation method and application of rabbit hemorrhagic disease virus VP10-VP60 recombinant protein
  • Rabbit hemorrhagic disease virus VP10-VP60 recombinant protein as well as preparation method and application of rabbit hemorrhagic disease virus VP10-VP60 recombinant protein
  • Rabbit hemorrhagic disease virus VP10-VP60 recombinant protein as well as preparation method and application of rabbit hemorrhagic disease virus VP10-VP60 recombinant protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 Construction of RHDV VP10-VP60 fusion protein gene expression vector

[0062] The RHDV VP60 gene was designed according to the protein sequence of NCBI Gene bank: DQ205345. The VP10 gene was designed according to the protein sequence of NCBI Gene bank: AJR27975.1.

[0063] The amino acid sequence of the VP10-VP60 recombinant protein fusion protein is as follows (SEQ ID NO.1):

[0064] MAFLMSEFVGLGLAGASVLSSALLRRQELQLQKQALENGLVLKANQLSRLGFNPNEVKSMIVGSNFSGNVKLSNMHNDASVVSAYSMYNPASNGIRKKIKSFNDSVKVYNTTGESSAMEGKTRTAPQGEAAGTATTASVPGTTTDGMDPGVVAATSVVTAENSSASVATAGIGGPPQQVDQQETWRTNFYYNDVFTWSVADAPGSILYTVQHSPQNNPFTAVLSQMYAGWAGGMQFRFIVAGSGVFGGRLVAAVIPPGIEIGPGLEVRQFPHVVIDARSLEPVTITMPDLRPNMYHPTGDPGLVPTLVLSVYNNLINPFGGSTNAIQVTVETRPSDDFEFVMIRAPSSKTVDSISPAGLLTTPVLTGVGNDNRWNGQIVGLQPVPGGFSTCNRHWNLNGSTYGWSSPRFADIDHRRGSASYSGNSSTNVLQFWYANAGSAIDNPISQVAPDGFPDMSFVPFNSPNIPTAGWVGFGGIWNSNNGAPAATTVQAYELGFATGAPNNLQPTTNTSGAQTVAKSIYAVVTGTNQNPTGLFVMASGVISTPNASAVTYTPQPDRIVTTPGTPAAAPVGKNTPIMFASVVRR...

Embodiment 2

[0069] Example 2 Expression of RHDV VP10-VP60 fusion protein

[0070]Transform the RHDV VP10-VP60 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, and pick a single clone colony , cultured in 300mL LB medium containing the same concentration of kanamycin at 37°C until the OD600 reached about 0.6, and induced expression with IPTG (Shanghai Sangong, product number: IB0168) with a final concentration of 1mM, the induction condition was: 25°C , 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 RHDV VP10-VP60 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 500w, 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, 0.3M 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 out, filtered through...

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Abstract

The invention discloses an RHDV VP10-VP60 recombinant protein, and belongs to the field of animal virus antibody detection. The recombinant protein comprises an amino acid sequence as shown in SEQ IDNO.1 or consists of an amino acid sequence as shown in SEQ ID NO.1. The invention also discloses a preparation method of the recombinant protein. The invention further discloses a gene of the recombinant protein, a vector containing the gene, a host cell containing the gene, a preparation method of the recombinant protein and application of the recombinant protein in an antibody for detecting RHDV. The recombinant protein disclosed by the invention is used for detecting the RHDV antibody, is convenient and rapid, has high sensitivity, does not have cross reaction with other pathogens, has strong specificity, and has huge clinical significance and wide application prospect.

Description

technical field [0001] The invention belongs to the field of animal virus antibody detection, and in particular relates to a recombinant protein of rabbit viral hemorrhagic disease virus VP10-VP60 and its preparation method and application. Background technique [0002] Rabbit haemorrhagic disease (Rabbit haemorrhagic disease, RHD) is also known as rabbit haemorrhagic pneumonia, rabbit haemorrhagic disease and rabbit plague. It is an acute, septic, highly contagious, lethal infectious disease of rabbits caused by rabbit viral hemorrhagic disease virus (RHDV). Systemic symptoms and even rapid death. [0003] Rabbit viral hemorrhagic disease has a rapid onset, violent spread, high morbidity and mortality rate. Once the rabbit group is infected with each other, the morbidity and mortality rate can be as high as 100%. It is one of the important rabbit diseases that seriously affect the healthy development of the rabbit industry. The disease occurs frequently in spring and summ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21G01N33/569C12R1/19
CPCC07K14/005C12N15/70G01N33/56983C12N2750/14022C12N2800/22C07K2319/00G01N2333/015G01N2469/20
Inventor 李晓光
Owner 杭州爱谨生物科技有限公司
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