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Monoclonal antibody of avian reticuloendotheliosis virus envelope protein and preparation method thereof

A monoclonal antibody, avian reticuloendothelial technology, applied in the intersection of molecular immunology and virology, can solve the problems of high price, false positives, false negatives, etc., and achieve the effect of large development potential

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

PCR technology is susceptible to interference from many factors, and false positive and false negative results may occur; the ELISA monoclonal antibody kits we currently use are produced abroad, which are expensive and costly to detect, which is not conducive to large-scale chicken detection and purification

Method used

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  • Monoclonal antibody of avian reticuloendotheliosis virus envelope protein and preparation method thereof
  • Monoclonal antibody of avian reticuloendotheliosis virus envelope protein and preparation method thereof
  • Monoclonal antibody of avian reticuloendotheliosis virus envelope protein and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1 Cloning and sequencing of REV env gene

[0066] A sequence at both ends of the env gene of the proviral genome DNA of the REV standard strain SNV strain was used as a primer. The upstream primer is 5'-CCGGAATTCGCCATCCTACAGAAAACAAA-3' whose gene sequence is shown in SEQ ID NO.2, and the downstream primer is 5'-CGACTCGA-GAACAATATGAGCCCAAACA-3' whose gene sequence is shown in SEQ ID NO.3, and in the primer EcoR I and Xhol I restriction sites were added, respectively. The PCR reaction conditions were: 95°C for 5 min, fully denatured and then entered the cycle system: 95°C for 30 s, 56°C for 1 min, 72°C for 2 min, a total of 30 cycles; then extended at 72°C for 10 min. The PCR products were identified by 1% agarose gel electrophoresis, and the results were as follows: figure 1 Purify PCR products using a DNA purification kit as indicated.

[0067] The pET-28a vector and PCR purified product were double-digested with restriction endonucleases EcoR I and Xhol...

Embodiment 2

[0070] Expression and purification of embodiment 2 His-env protein

[0071] Inoculate the BL21 bacteria containing the recombinant plasmid in the LB solid medium containing kanamycin, culture at 37°C, pick a single colony and inoculate it in the LB liquid medium containing Kan (final concentration: 30 μg / ml), shake at 37°C (200rpm), cultivated to OD600=0.6, added IPTG to a final concentration of 0.4mmol / L, continued to cultivate at 37°C for 3h, and set BL21 bacteria transformed with uninduced recombinant plasmids as a control.

[0072] SDS-PAGE analysis showed that the target protein His-env was expressed in the form of inclusion bodies, and the detection steps were as follows:

[0073] 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...

Embodiment 3

[0099] Example 3 animal immunity

[0100] The above-mentioned purified His-env fusion protein was used to immunize 6-week-old Balb / C female mice, the immunization dose was 40ug each time, and the immunization method was intraperitoneal injection, and immunized four times in total.

[0101] 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 adjuvant w...

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Abstract

The invention belongs to the cross technical field of molecular immunology and virology, and particularly relates to a monoclonal antibody of avian reticuloendotheliosis virus envelope protein. The monoclonal antibody is characterized in that 1200bp of genetic fragment in a relative conservation region of an avian reticuloendotheliosis virus (REV) envelope (env) gene is selected, and 400 amino acids expressed by the fragment comprise more than 90% of antigen sites of the REV, so that most of the antigen sites of the virus are retained and the interference caused by genovariation is excluded, prokaryotic expression product His-env fusion protein is obtained by utilizing the env gene containing the conservation genetic fragment, the Balb / C mouse is immunized by the fusion protein, and the monoclonal antibody of the REV ENV (envelope) protein is obtained through fusion of oncocyte SP2 / 0 and splenocyte of the immunized mouse, screening and cloning. The monoclonal antibody provides a technical support for diagnosis and prevention as well as scientific research of avian reticuloendotheliosis.

Description

Technical field [0001] The invention involves the field of molecular immunology and virus crossover technology, which specifically involves a monoclonal antibody and its preparation method of a conservative sequence of poultry mesh endothelial tissue hyperplasia virus cyst protein (ENV) secreted by hybrid tumor cells. Background technique [0002] Poultry mesh endothelial hyperplasia (RE) is a group of syndrome (Witter R. L, 1997), which is characterized by mesh cell hyperplasia (REV), which is mainly characterized by mesh cell hyperplasia (REV).Chronic tumor hyperplasia of the chronic tumor hyperplasia, short composite syndrome and lymphatic tissue and other tissues can cause severe growth inhibitory and immunity.Following the poultry Malik's disease and poultry leukemia, RE is the third infectious tumor disease that has been determined to date. [0003] In addition to knowing that there are different REV strains such as turkey, chicken, duck, goose, Japanese quail, etc., they a...

Claims

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Application Information

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IPC IPC(8): C07K16/08C12N5/20C12N15/34C12N15/63C12R1/91
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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