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Z structural domain derivative of staphylococcus protein A and application thereof

A Staphylococcus, structural domain technology, applied in the direction of anti-bacterial immunoglobulin, application, peptide/protein composition, etc., can solve problems such as recurrence, reduction of specific protein A antibodies, and pathogenic bacteria infection

Active Publication Date: 2015-12-30
浓孚雨医药河北有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, due to the strong affinity between the IgG Fc segment and protein A, it can compete with phagocytes, reduce the body's production of specific protein A antibodies, and lead to the infection and recurrence of pathogenic bacteria in the human body.

Method used

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  • Z structural domain derivative of staphylococcus protein A and application thereof
  • Z structural domain derivative of staphylococcus protein A and application thereof
  • Z structural domain derivative of staphylococcus protein A and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0180] The construction of embodiment 1 protein carrier

[0181] According to the sequence of the open reading frame of the sumo gene (a small molecule ubiquitin-like modified protein, used as a fusion tag for recombinant protein expression), the T domain gene mRNA of DT and the protein A gene mRNA in GeneBank, the primers were designed by the principle of overlapping PCR in After the sumo gene, connect the T domain of DT, and then introduce one of the domain Z of protein A, and introduce two mutations in domain Z, F13G / Y14G, and introduce restriction sites NdeI and BamHI at the head and tail , and the introduction of protective bases, connected to PET28. Synthesized by Nanjing GenScript Biotechnology Co., Ltd. according to the primers. The peptide chain amino acid sequence of the specifically designed sumo-DTT-ZFY of the present invention is shown in SEQIDNO: 7; its DNA sequence is shown in SEQIDNO: 8; and the primers are designed and synthesized as follows: P1: SEQIDNO: 14;...

Embodiment 2

[0197] Example 2 Expression identification and large-scale preparation of protein

[0198] Protein

[0199] A single colony of Escherichia coli containing the recombinant expression plasmid sumo-DTT-ZFY fusion protein was inoculated into 5 mL of LB medium and cultured overnight at 37°C. Then diluted 1:50 into 50 mL of LB medium containing 30 ng / L kanamycin, cultured for about 4 hours to make the OD600 value about 0.6, added IPTG to a final concentration of 1 mM, and took out after induction at 37°C for 4 hours sample.

[0200] Take 50mL of the induced culture, place it on ice for 30min, centrifuge at 12000rpm for 8min, collect the bacteria, discard the supernatant; dissolve and resuspend the precipitate with 1×PBS (5mL / 100mL culture) plus an equal volume of ice-cold sterile water; add 300μg / mL lysozyme, shake gently on ice for 30min, then freeze at -70°C for 12h; melt the sample with running water, add protease inhibitors, ultrasonically break (200W power) in an ice bath, br...

Embodiment 3

[0207] Embodiment 3 isothermal titration experiment

[0208] By adopting isothermal titration calorimetry method, the change of the binding constant of Z domain of native protein A and ZFY mutant to human and rabbit IgG was determined. If it is necessary to verify that the mutated domain ZFY has almost no binding reaction with IgG, it can be intuitively reflected by the change of its reaction energy. If there is no obvious energy change during the mixing process, it can be considered that there is no binding between the ZFY protein and IgG, and the point mutation transformation of the Z domain is successful; and if the two have obvious energy changes during the mixing process If the energy changes, it can be inferred that the ZFY protein will still react with IgG, and such point mutations have no effect or are incomplete. Through isothermal titration experiments, it is possible to effectively verify whether the transformation of the Z domain of protein A is successful in vitr...

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Abstract

The invention discloses a Z structural domain derivative of staphylococcus protein A and application thereof. Particularly, the provided Z structural domain derivative of staphylococcus protein A has the capacity for combining IgG, Z structural domain amino acid is mutated to form the Z structural domain derivative, and KZFY / KZ<=10%, wherein KZFY is the combining constant of the Z structural domain derivative and IgG, and KZ is the combining constant of a Z structural domain and IgG; after the Z structural domain derivative is used for immunizing animals, the animals can be induced to generate immunoreaction for staphylococcus protein A. The ZFY derivative and carrier protein DTT are coupled to obtain a kind of protein which can be expressed and purified conveniently, and the protein can effectively stimulate an immune system to generate protein A antibodies.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular, the invention relates to a derivative of staphylococcus protein A and its application. Background technique [0002] Staphylococcus aureus (Staphylococcus aureus, hereinafter referred to as Staphylococcus aureus) is one of the main pathogens of hospital and community-acquired infections, which can cause human suppurative infections, skin and soft tissue infections, necrotizing pneumonia, sepsis, bacteremia disease, osteomyelitis and endocarditis. At present, Staphylococcus aureus with multiple antibiotic resistance has brought great difficulties to the treatment, especially the rapid spread of methicillin-resistant strains has made it more and more difficult to rely solely on antibiotics to treat related diseases caused by Staphylococcus aureus. The less reliable. Immunotherapy can activate or enhance the body's immune system, act synergistically with antibiotics, reduce the selection ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/31C07K19/00C07K16/12C12N15/31C12N15/62C12N15/63A61K38/16A61K48/00A61K39/085A61P31/04
Inventor 李荣秀李灵舒
Owner 浓孚雨医药河北有限公司
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