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Babesia mocroti Bm7 recombinant antigenic protein, preparation method and purpose thereof

A technology of recombinant antigenic protein and recombinant protein, applied in cell biology research and molecular fields, can solve the difficulty of morphological diagnosis of Babesia, the situation of Babesia infection and carriers is unclear, and Babesia vole does not appear Issues such as the public report on the diagnosis of babesia with Bm7 recombinant antigen protein

Inactive Publication Date: 2015-07-08
STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the natural foci of Babesia are widely distributed across the country and cases are reported from time to time, the situation of Babesia infection and carriers in these high-risk areas is still unclear
[0004] (2) The occult nature of Babesia infection is widespread: Babesia is opportunistically pathogenic, and Babesia infection in the population can range from asymptomatic to Babesia-infected immunodeficiency, such as HIV-infected patients or low-level patients with obvious clinical symptoms, Even death, many asymptomatic carriers are detected to transmit pathogens to immunocompromised blood or organ receptors through blood donation or organs, resulting in babesiosis, so Babesia infection is hidden in healthy people, threatening blood product safety
At present, there are no screening standards and tools for blood donors with recessive infection of Babesia in my country, and there is an urgent need for screening methods with high sensitivity and specificity to ensure the safety of blood transfusion and related blood products.
[0005] (3) Difficulty in morphological diagnosis of Babesia: Babesia merozoites, especially B.microti, are similar in morphology to those of Plasmodium falciparum (Plasmodium falciparum) at the parasitic erythrocyte stage
[0009] Before the present invention, there was no public report related to the Babesia microti Bm7 recombinant antigen protein of the present invention and its use in the diagnosis of Babesia

Method used

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  • Babesia mocroti Bm7 recombinant antigenic protein, preparation method and purpose thereof
  • Babesia mocroti Bm7 recombinant antigenic protein, preparation method and purpose thereof
  • Babesia mocroti Bm7 recombinant antigenic protein, preparation method and purpose thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Cloning of embodiment 1 Babesia mori Bm7 gene

[0055] 1.1 Amplification and purification of Bm7 gene fragments:

[0056] 1.1.1 According to the sequence of Bm7 gene (Genbank CCF73510.1) (sequence shown in SEQ ID NO.1), utilize PrimerPremier5.0 software to design a pair of specific primers, as follows:

[0057] PF: 5'-CCG GAATTC ATGCATATCAACTACAAATTAATTATA-3' (as shown in SEQ ID NO.3);

[0058] PR: 5'-CCG CTCGAG TTAAGCAGCATTAGGTGTG-3' (as shown in SEQ ID NO.4);

[0059] The parts in italics are the protective bases of the upstream and downstream primers, and the bold parts are the EcoR I restriction sites of the upstream primers and the Xho I restriction sites of the downstream primers. Specific primers were synthesized by Yingwei Jieji (Shanghai) Trading Co., Ltd.

[0060] 1.1.2 Using Babesia vole cDNA as template, carry out PCR reaction to amplify Bm7 gene, the reaction system is as follows:

[0061]

[0062] Reaction conditions:

[0063]

[0064] 1.2% agar...

Embodiment 2

[0077] Example 2 Construction of Bm7 Gene Recombination Plasmid pET-28a(+)-Bm7

[0078] 2.1 Double digestion and recovery of PCR products

[0079] The recovered PCR target fragment was double-digested overnight in a water bath at 37°C. The enzyme digestion reaction system was as follows:

[0080]

[0081] Digested products were subjected to 1.2% agarose gel (GoldView TMNucleic acid dye) electrophoresis, cut off the target band, and recover the DNA molecule again, the recovery method is the same as 1.1.3 in Example 1, and the recovered product is stored at -20°C.

[0082] 2.2 Preparation of pET-28a(+) Empty Plasmid (AxyPrep Plasmid Miniprep Kit)

[0083] Pick a single colony containing the pET-28a (+) plasmid on an LB plate (containing 50 μg / ml kanamycin) and culture it overnight at 37° C. in 5 ml LB medium (containing 50 μg / ml kanamycin). On the next day, transfer 1ml of the culture solution into a 1.5ml centrifuge tube and store at 4°C as a strain. The remaining cultur...

Embodiment 3

[0092] Example 3 Identification of Babesia microti Bm7 expression vector

[0093] 3.1 Ligation product transformed into E.coli DH5α competent cells

[0094] Gently mix the ligation product in 2.4 of Example 2 with E.coli DH5α competent cells, ice-bath for 30 minutes, heat shock in a water bath at 42°C for 1.5 minutes, and ice-bath again for 5 minutes. Add 900 μl of SOC medium to the culture tube under sterile conditions, shake at 37° C. and 200 rpm for 1 hour. Centrifuge the cultured bacterial solution at 3500 rpm for 3 minutes, discard most of the supernatant, and leave about 100 μl of medium to resuspend the bacterial cells. The resuspension was evenly spread on LB plates (containing 50 μg / ml kanamycin), cultured upside down at 37° C. overnight, and the growth of colonies was observed.

[0095] 3.2 PCR identification of recombinant plasmids

[0096] A single colony on the plate was randomly selected for colony PCR identification, and the PCR product was detected by agaros...

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Abstract

The invention discloses a babesia mocroti Bm7 recombinant antigenic protein, which comprises protein with an amino acid sequence shown in a SEQ ID NO.2, or a protein with the same functions formed by replacing and deleting or inserting the protein. In addition, the invention also discloses a preparation method of the babesia mocroti Bm7 recombinant antigenic protein, which comprises the steps of babesia mocroti Bm7 gene sequence amplification, babesia mocroti Bm7 recombinant plasmid construction and identification, and inducible expression and purifying of the recombinant protein. In addition, the invention also discloses an application of babesia mocroti Bm7 recombinant antigenic protein in preparation of a reagent or a kit for detecting babesia mocroti disease patients (animal or human) serum antibody. Through verification, the babesia mocroti Bm7 recombinant antigenic protein used for babesia mocroti diagnosis has high sensitivity and singularity, is latent diagnosis candidate target, and can be taken as a target antigen for babesia mocroti disease diagnosis.

Description

technical field [0001] The invention relates to the field of molecular and cell biology research, in particular to a recombinant antigenic protein of Babesia microti unnamed protein (Bm7) and a preparation method thereof. In addition, the present invention also relates to the application of the Babesia microti Bm7 recombinant antigen protein in the preparation of reagents or kits for detecting serum antibodies of Babesia microti patients. Background technique [0002] Babesia is an important tick-borne protozoan that parasitizes in the red blood cells of humans or other mammals. It can cause zoonotic babesiosis and is distributed in Asia, Europe, America, and Africa. Babesia is opportunistically pathogenic, and its pathogenicity is enhanced for immunocompromised persons, and even life-threatening for immunocompromised persons. As a zoonotic tick-borne parasitic disease, the research of Babesia in my country is mainly in the field of animal husbandry and veterinary medicine....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/44C12N15/70C12N15/30C12N15/11G01N33/68
CPCC07K14/44C12N15/11C12N15/70G01N33/68
Inventor 胡薇孙嘉慧徐斌周霞鞠川陈军虎沈海默黄继磊张铤莫筱瑾陈绅波周晓农
Owner STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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