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Human L. pneumophila surface protein monoclonal antibody and antigen capture ELISA kit

A technology of Legionella pneumophila and monoclonal antibody, applied in the field of immunology, can solve the problem that the role of outer membrane lipoprotein is not clear enough

Active Publication Date: 2019-12-06
湖北云璐生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the role of this outer membrane lipoprotein in the pathogenesis of Legionnaires' disease is not yet clear.

Method used

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  • Human L. pneumophila surface protein monoclonal antibody and antigen capture ELISA kit
  • Human L. pneumophila surface protein monoclonal antibody and antigen capture ELISA kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Preparation of recombinant human Legionella pneumophila surface protein PAL

[0035] 1) Cloning and expression of human Legionella pneumophila PAL gene

[0036]Bioinformatic analysis of the human Legionella pneumophila surface protein PAL (the accession number in the NCBI protein database is YP_096059) gene, combined with GC content, codon bias, mRNA secondary structure, RNA instability motif, mRNA freedom Considering performance stability, etc., the DNA coding sequence was optimized, and at the same time, the whole gene sequence was chemically synthesized after introducing the restriction site NdeI at the 5' end, the termination signal TAA and the restriction site EcoRI at the 3' end (the whole sequence synthesis was handed over to Kim Completed by Sirui Biotechnology Co., Ltd., the artificially synthesized gene fragment was connected to the vector pUC57 at the time of delivery), denoted as PAL'. The full sequence of the gene and the encoded amino acid seque...

Embodiment 2

[0040] Example 2 Preparation of Human Legionella Pneumophila Surface Protein Polyclonal Antibody

[0041] 1) Immunization of New Zealand purebred rabbits

[0042] The recombinant PAL protein prepared in Example 1 was mixed with complete Freund's adjuvant, emulsified and used as an immunogen to immunize 2 male New Zealand rabbits, each rabbit was subcutaneously injected with a total amount of 2ml, and the total amount of antigen was 2mg / only. Afterwards, the emulsion formed by recombinant PAL protein and Freund's incomplete adjuvant was used to immunize once every two weeks, for a total of 5 times, and the amount of antigen was the same as that of the first immunization. 3-5 days after the fifth immunization, a large amount of blood was taken from the heart, placed at 37°C for 1 hour, then placed in the refrigerator at 4°C overnight, and serum was collected the next day.

[0043] 2) Determination of polyclonal antibody titer

[0044] Recombinant PAL protein was used as the co...

Embodiment 3

[0052] The preparation of embodiment 3 human Legionella pneumophila surface protein monoclonal antibody Lp-2#

[0053] 1) Immunization of animals

[0054] Using the recombinant human Legionella pneumophila surface protein PAL prepared in Example 1 as an antigen, 5 8-week-old BALB / c mice were immunized, and 2 were not used as immunized mice as negative controls. After the initial immunization antigen was fully emulsified with an equal amount of Freund's complete adjuvant, the mice were subcutaneously injected into the back of the immunized mice at a rate of 100 μg / mouse. After that, the second immunization was performed by intraperitoneal injection with the same dose of antigen fully emulsified with Freund's incomplete adjuvant at an interval of three weeks, and the third time was injected intraperitoneally with the same dose of antigen fully emulsified with Freund's incomplete adjuvant at an interval of 2 weeks. immunity. Blood was collected from the tail vein 15 days after ...

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Abstract

The invention discloses a human L. pneumophila surface protein monoclonal antibody and an antigen capture ELISA kit. The human L. pneumophila surface protein monoclonal antibody is generated by secreting of hybridoma cell strains with the preserving number being CCTCCNO: C2017216. The human L. pneumophila surface protein monoclonal antibody can be used for detecting human L. pneumophila. The invention further discloses the human L. pneumophila surface protein capture ELISA kit based on the monoclonal antibody.

Description

technical field [0001] The invention belongs to the field of immunology and relates to a human legionella pneumophila surface protein monoclonal antibody, a hybridoma cell line and an antigen capture ELISA kit. Background technique [0002] Legionella is a Gram-negative pathogen that often pollutes water environments and causes acute respiratory infections in humans, and can be used as a biological warfare agent. Legionella is an aerobic Gram-negative bacillus with a sterile membrane that does not produce acid or gas. It has one or several terminal flagella or lateral flagella and can move. At present, 49 species and more than 70 serotypes of Legionella have been found, at least 20 of which are related to human diseases. The common ones are Legionella pneumophila (L.pneumophila, LP), Legionella majestica, Legionella burgdorferi, Legionella fischeri, Legionella dunai, Legionella longbeach, etc. Among them, Legionella pneumophila is the cause of acquired pneumonia. important...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/12C07K14/195C12N15/31C12N15/70G01N33/569G01N33/543G01N33/577
CPCC07K14/195C07K16/1203C07K2317/35C12N15/70G01N33/54306G01N33/56911G01N33/577G01N2469/10
Inventor 胡征
Owner 湖北云璐生物工程有限公司
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