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Anti-interferon alpha-2b nanobody and application thereof

A nanobody, anti-interferon technology, applied in the direction of interferon, anti-cytokine/lymphokine/interferon immunoglobulin, application, etc., can solve the large demand for interferon α-2b and the cumbersome production process of monoclonal antibody , polyclonal antibody specificity is not high, to achieve the effect of small molecular weight, high specificity, good binding specificity

Active Publication Date: 2018-03-30
PEKING UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are three subtypes of genetically engineered interferon on the market in my country: α-1b, α-2a, and α-2b. Among them, the purified and quality-tested interferon α-2b on the market are all monoclonal or polyclonal antibodies. Due to the tedious and complicated production process of monoclonal antibodies, the stability is poor; the specificity of polyclonal antibodies is neither high nor stable, and the market demand for interferon α-2b is large

Method used

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  • Anti-interferon alpha-2b nanobody and application thereof
  • Anti-interferon alpha-2b nanobody and application thereof
  • Anti-interferon alpha-2b nanobody and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Example 1: Panning and Identification of Anti-Interferon α-2b Nanobodies

[0028] The reagents and materials used in the present invention are all commercially available. Among them, anti-interferon α-2b was purchased from China National Institutes for Food and Drug Control, and the phage display library was purchased from Nanchang University.

[0029] 1. Phage library panning

[0030] (1) Coating: Dissolve the antigen in PBS, add to a microtiter plate (100 μL / well), and coat overnight at 4°C.

[0031] (2) Blocking: Aspirate the coating solution, wash the plate three times with PBS, and incubate with 300 μL of 3% BSA-PBS at 37° C. for 2 h.

[0032] (3) Binding: Aspirate the blocking solution, wash the plate 3 times with PBS, add 100 μL phage display library (immune library of interferon α-2b, alpaca species)

[0033] (Optional step: titer to 2×10 12 cfu / mL, mixed with an equal volume of BSA-PBS solution, pre-incubated at 20-30°C for 1h), and incubated at 37°C for ...

Embodiment 2

[0076] Example 2: Expression and purification of Nanobodies

[0077] Take 2ul of the pET28a vector constructed by Purple and add it to 50ul of BL21 competent, place it on ice for 30 minutes, then place it in a water bath at 42°C for 90 seconds, place it on ice for 5 minutes, and then add 600ul of LB The medium was cultured in a 37°C incubator for 1 hour; then 100ul of the above culture solution was applied to an ampicillin plate, and placed in a 37°C incubator for overnight culture. The next day, single clones were picked from the plate and inoculated into 1L culture medium for cultivation at 37°C and 220rmp. When the bacterial solution OD=0.5, add IPTG with a final concentration of 0.1mM and induce overnight at 16°C and 180rmp. The next day, centrifuge the bacterial liquid and resuspend the bacterial cells in 50ml PBS, and then perform ultrasonic crushing. The ultrasonic condition is 200w, crushing for 3 seconds, with an interval of 3 seconds; The nanobody is adsorbed on t...

Embodiment 3

[0078] Example 3: Western blot verification

[0079] Take 1ug of rhIFN-α2b, 2ug of rhIFN-α2b and 1ug of MG53 protein as samples respectively, add 5ug of I22 antibody, incubate for 1h and add anti-HIS tag secondary antibody, see the experimental results Figure 4 .

[0080] Depend on Figure 4 It can be seen that rhIFN-α2b has a clear band, and there is no band at MG53, which proves that the Nanobody I22 of the present invention can specifically bind to the IFN-α2b protein.

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Abstract

The invention discloses an anti-interferon alpha-2b nanobody and application thereof. The anti-interferon alpha-2b nanobody comprises one or two VHH chains, wherein the VHH chain consists of frameworkregions and complementary determining regions, wherein the complementary determining regions consist of CDR1 as shown in SEQ ID NO:1, CDR2 as shown in SEQ ID NO:2 and CDR3 as shown in SEQ ID NO:3; and the framework regions consist of FR1 as shown in SEQ ID NO:4, FR2 as shown in SEQ ID NO:5, FR3 as shown in SEQ ID NO:6 and FR4 as shown in SEQ ID NO:7. The invention also comprises application of the anti-interferon alpha-2b nanobody. According to the anti-interferon alpha-2b nanobody and the application thereof, the anti-interferon alpha-2b nanobody has the advantages of high stability, high specificity, small molecular weight, and being suitable for large-scale production; and can be made into a purification column for the purification of the interferon alpha-2b, and can also be made intoa secondary antibody reagent for the detection of the interferon alpha-2b, so that the anti-interferon alpha-2b nanobody has a broad application prospect.

Description

technical field [0001] The present invention relates to a nanobody and its application, in particular to an anti-interferon alpha-2b nanobody and its application. Background technique [0002] Since the advent of hybridoma technology, monoclonal antibodies have been widely used in the diagnosis and treatment of various diseases. However, these monoclonal antibodies mainly derived from mice are large in size and poor in stability, which limits their clinical application. [0003] Nanobodies, also known as single domain heavy chain antibodies or VHH antibodies, are antibodies that naturally exist in camels without light chains. Compared with ordinary antibodies and recombinant single chain fragment variable (single chain fragment variable, scFv), nanobodies have suitable It can be transformed into various forms through genetic modification, so it has broad application prospects. [0004] Interferon (IFN) is a broad-spectrum antiviral agent. It does not directly kill or inhib...

Claims

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Application Information

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IPC IPC(8): C07K16/24C07K14/56C07K1/22C12N15/13C12P21/02G01N33/68
CPCC07K14/56C07K16/249C07K2317/565C07K2317/567C07K2317/569C07K2317/92G01N33/6866
Inventor 林坚周鹏秦玺饶春明裴德宁李承鹏段茂琴徐良代青松周斌
Owner PEKING UNIV
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