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Monoclonal antibody of avian leukosis virus subgroup J surface protein and preparation method thereof

A technology of avian leukosis virus and monoclonal antibody, which is applied in the interdisciplinary field of molecular immunology and virology, can solve problems such as false positives, false negatives, and inaccurate test results, and achieve low-cost results

Inactive Publication Date: 2012-01-04
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The PCR method is susceptible to interference from many factors, and false positive and false negative results may occur; ELISA monoclonal antibody kits are imported from abroad. Due to the highly variable ALV-J gp85 gene from different sources, imported detection kits are not suitable for the detection of domestic strains. Inconclusive; immunohistochemistry and indirect immunofluorescence also require highly specific monoclonal antibodies

Method used

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  • Monoclonal antibody of avian leukosis virus subgroup J surface protein and preparation method thereof
  • Monoclonal antibody of avian leukosis virus subgroup J surface protein and preparation method thereof
  • Monoclonal antibody of avian leukosis virus subgroup J surface protein and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Cloning and sequencing of ALV-J gp85 gene

[0072] The sequences at both ends of the envelope glycoprotein gene of ALV-J Chinese strain WS0705 provirus genome DNA gp85 were used as primers. The forward primer is 5'-CTGGATCCATGGGAGTTCATCTATTGCAACACCCAG-3', as shown in SEQ ID NO.2, containing the BamHI restriction site; the reverse primer is 5'-TACTGCAGT TAG CGC CTG CTA CGG TGG TGA CC-3', as shown in SEQ ID NO. .3, containing PstI enzyme cutting site. The PCR reaction conditions were: 95°C for 4 min, fully denatured and then entered the cycle system: 95°C for 30 s, 59°C for 1 min, 72°C for 2 min, a total of 30 cycles; then extended at 72°C for 10 min. The PCR product was purified using a DNA purification kit, and the length of the PCR product was detected to be 924 base pairs by 1% agarose gel electrophoresis. figure 1 As shown, its gene sequence is shown in SEQ ID NO.1, and the PCR product was purified using a DNA purification kit.

[0073] The pProEXHTb vector and...

Embodiment 2

[0077] Expression and purification of His-gp85 protein

[0078] Inoculate the BL21 bacteria containing the recombinant plasmid in the LB solid medium containing ampicillin (Amp), cultivate at 37°C, pick a single colony and inoculate it in the LB liquid medium containing Amp (final concentration: 50 μg / ml), at 37°C Shake (200rpm), cultivate to OD600=0.6, add IPTG to a final concentration of 1mmol / L, continue to cultivate at 37°C for 3h, and set BL21 bacteria transformed with uninduced recombinant plasmids as a control.

[0079] SDS-PAGE analysis showed that the target protein His-gp85 was expressed in the form of inclusion bodies, and the detection steps were as follows:

[0080] Collect the cultivated engineering bacteria liquid, centrifuge at 5000rpm for 5min, and discard the supernatant; weigh 30g of the collected wet bacteria into a 500ml beaker, add 300ml of resuspension, add the rotor and stir on a magnetic stirrer for 20min to make the bacteria Disperse evenly; place ...

Embodiment 3

[0107] animal immunity

[0108] The 6-week-old Balb / C female mice were immunized with the above-mentioned purified His-gp85 fusion protein, the immunization dose was 40ug per mouse, and the immunization method was intraperitoneal injection, and the immunization was done four times in total.

[0109] For the first immunization, use the above protein to emulsify the antigen with the same amount of Freund's complete adjuvant, and perform the second immunization two weeks later. During the immunization, emulsify the inactivated antigen with the same amount of Freund's incomplete adjuvant, and perform three immunizations two weeks later , The immunization method and dose are the same as the second immunization. One week after the three times of immunization, blood was collected from the orbit of the mice to measure its titer, and the serum antibody titer was monitored. Two weeks after the third immunization, the mouse with the highest titer was selected, and the antigen without ...

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Abstract

The invention belongs to the cross technical field of molecular immunology and virology, and particularly relates to a hybridoma cell based on hybridoma type leukosis virus subgroup J isolated strain with preservation number of CGMCC (China General Microbiological Culture Collection) No. 5018, a high-specificity monoclonal antibody of avian leukosis virus subgroup J (ALV-J) surface protein (SU) secreted by the hybridoma cell and a preparation method of the monoclonal antibody. By using the high-specificity monoclonal antibody prepared by the invention, a technical support is provided for diagnosis, prevention and treatment of avian leukosis virus subgroup J disease, and the economic loss caused by avian leukosis virus subgroup J disease is effectively reduced.

Description

technical field [0001] The present invention relates to the technical fields of molecular immunology and virology, in particular to a hybridoma cell based on an angiomatous J subgroup leukemia strain isolated from a case, and the J subgroup avian leukemia virus (ALV) secreted by the hybridoma cell. -J) Highly specific monoclonal antibody to surface protein (SU) and its preparation method. Background technique [0002] Subgroup J avian leukemia is a neoplastic disease caused by subgroup J avian leukemia virus (ALV-J). Clinical infection is mainly manifested as the induction of various tumors, immune tolerance, and decreased production performance. Among the natural cases, avian lymphocytic leukemia is the most common, which is the most harmful to the poultry industry. [0003] 1. The new characteristics of J subgroup avian leukemia epidemic in chickens in my country [0004] In recent years, subgroup J avian leukemia has shown an outbreak trend in chicken flocks in my coun...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N5/20
Inventor 成子强王峰张利王桂花王玥于琳琳姜艳萍王晓伟陈洪博
Owner SHANDONG AGRICULTURAL UNIVERSITY
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