A kind of recombinant Lawsonia intracellularis porcine hsp60 protein monoclonal antibody and its application

A monoclonal antibody, Lawsonia technology, applied in anti-bacterial immunoglobulin, application, immunoglobulin and other directions, can solve the problem of less research, achieve small molecular weight, high specificity, suitable for commercial large-scale production. Effect

Active Publication Date: 2022-04-22
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

LI Hsp60 protein is a molecular chaperone protein of Lawsonia intracellulare, which is rarely studied in LI. So far, there is no relevant report on the preparation of monoclonal antibodies to Lawsonia intracellulare Hsp60 protein at home and abroad

Method used

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  • A kind of recombinant Lawsonia intracellularis porcine hsp60 protein monoclonal antibody and its application
  • A kind of recombinant Lawsonia intracellularis porcine hsp60 protein monoclonal antibody and its application
  • A kind of recombinant Lawsonia intracellularis porcine hsp60 protein monoclonal antibody and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0063] Example 1: Expression and purification of Lawsonia intracellulare Hsp60 protein

[0064] 1) PCR amplification of Hsp60 gene

[0065] According to the Hsp60 gene sequence of Lawsonia intracellulare in GenBank, DNAStar biological software was used to analyze, select the region with good antigenicity, and entrust Shanghai Sangon Bioengineering Co., Ltd. to synthesize a Hsp60 gene with EcoR I and Sal I restriction sites Sequence, the expected target fragment length is 1647bp, the Hsp60 gene-specific primer sequence is F1 5'CG-GAATTC-ATGGCTTCTAAAGAAATC3'; R15'GC-GTCGAC-CTAGTACATACC GTCCAT 3' (the underlines indicate EcoR I and Sal I restriction sites respectively) . The 25 μL PCR reaction system is: 2 μL of ileitis-positive pig fecal DNA, 12.5 μL of Mix, 1 μL of 10 μmol / L upstream and downstream primers, ddH 2 O 8.5 μL. PCR amplification conditions were: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C fo...

Embodiment 2

[0075] Example 2: Preparation of monoclonal antibody against Lawsonia intracellulare Hsp60 protein

[0076] 1) Immunogen preparation and animal immunization procedures

[0077] The purified Hsp60 protein was mixed with complete Freund's adjuvant at a ratio of 1:1, and emulsified into an immunogen. The immunogen was immunized with 7-week-old Bab / C mice by multi-point subcutaneous injection on the back. waived. Ten days after the third immunization, blood was collected from the orbital venous plexus of the mice, the serum was separated, and the antibody level in the serum was determined by indirect ELISA. The mouse with the highest antibody level was selected for shock immunization 3 days before fusion, that is, the purified Hsp60 protein was directly injected intraperitoneally without adding any adjuvant to the immunogen, 50 μg / mouse.

[0078] 2) cell fusion

[0079] Preparation of feeder cells

[0080] (1) 8-week-old Bab / C mice were sacrificed by cervical dislocation and ...

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Abstract

The invention discloses a recombinant Lawsonia intracellularis Hsp60 protein monoclonal antibody and its application. The heavy chain variable region of the monoclonal antibody includes HCDR1, HCDR2 and HCDR3, and its amino acid sequences are respectively shown in SEQ ID NO.1 , shown in SEQ ID NO.2 and SEQ ID NO.3; the light chain variable region of the monoclonal antibody includes LCDR1, LCDR2 and LCDR3, and its amino acid sequences are as shown in SEQ ID NO.4, SEQ ID NO.5 and Shown in SEQ ID NO.6. The recombinant Lawsonia intracellularis suis Hsp60 protein monoclonal antibody prepared by the present invention has high purity, good specificity and high sensitivity, and has no cross-reactivity with other intestinal pathogenic bacteria, can be used for ELISA and IFA detection, and is useful for clinical LI diagnosis It is of great significance to research and develop laboratory LI diagnostic technology.

Description

technical field [0001] The invention belongs to the technical field of monoclonal antibodies, and in particular relates to a recombinant Lawsonia intracellularis Hsp60 protein monoclonal antibody and application thereof. Background technique [0002] Lawsonia intracellularis (LI) is an important intestinal pathogen, which mainly causes clinical symptoms such as acute hemorrhagic diarrhea, intermittent diarrhea, loss of appetite and slow growth and development in growing and finishing pigs aged 6 to 20 weeks. The loss caused by ileitis reaches 100 million U.S. dollars per year, and subclinical ileitis will cause economic losses of 4 U.S. dollars per pig, bringing significant economic losses to the pig industry. It has become an important intestinal disease in modern pig industry. It is ubiquitous in pig farms all over the world, and it is more serious in developing countries. A survey of fattening pigs in 54 pig farms in China found that the positive rate of this disease acc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/12C12N15/13G01N33/577G01N33/569
CPCC07K16/12G01N33/577G01N33/56911C07K2317/56C07K2317/565G01N2333/195G01N2469/10
Inventor 范红结李剑男肖宁胡雨婷周红蔺辉星陈长风
Owner NANJING AGRICULTURAL UNIVERSITY
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