Amplification And Expression Of IgY Antibody Gene Against Avian Infectious Bronchitis Virus

This experiment is to use genetic engineering antibody technique was used to prepare chicken infectious bronchitis IgY antibodies, for chicken antibody gene research and specific antibody preparation to provide relevant data, as well as treatment and diagnosis of chicken infectious bronchitis to offer help.In order to obtain the full antibody gene sequence of chicken IgY. In this research, we extracted the total RNA from Chicken’s spleen then as a template reverse transcribed into cDNA, according to the published light chain sequence designed primers with PCR amplified light chain ORF sequence. Due to the lack of online published antibody heavy chain of the full gene sequence, we according to the published heavy chain constant region sequence designed primers, respectively using the 5’RACE and 3’RACE amplification, will splicing the correct two sequence by DNAStar software, found the ORF sequence, re-designing the primer to amplify the heavy chain sequence. Band of amplification of Light and heavy chain were connected respectively with cloning vector pMD19-T, transfecting competent cells DH5a to pick positive clones, it has been verified by enzyme digestion and PCR correct plasmid sequenced. Sequence analysis showed that the amplification of light chain ORF sequences is 705bp, encoding 234 amino acids with the published sequence homology of 95%; heavy chain ORF sequence has 1716 bp, encoding 571 amino acids, and its variable region gene sequence in chicken antibody heavy chain matches variable region characteristics, its constant region sequence with published chicken IgY antibody heavy chain gene sequence share homology of 99%.We have successfully built gene amplification chicken IgY antibodies light and heavy chain sequencing method.In order to express the chicken IBV specified antibody gene sequence, let 30 1-day-old chicks were randomly divided into 2 groups, respectively, raised in isolation room. After maternal antibody level dropped, the first group were immunized with attenuated vaccine strain 4/91 and the second immunization was implemented after 14 days, the second remained untreated as blank control.Every seven days after the first immunization, serum was separated from the blood collected from the 2 groups of chickens. Indirect ELISA was carried out to detect specific antibodies. Spleens were collected for RNA extraction to amplify antibody gene when the antibody level significantly increased.Variable region of light chain was found to vary in a wide range but small variation happened to heavy chain. An antibody gene selected from immunized chicks were directed cloned into eukaryotic vector pcDNA3.1, reconstructing recombination expression vector pcDNA3.1-H and pcDNA3.1-L. Two recombined vectors which have been correctly sequenced were co-transfected 293T cell. After 48 hours, together with indirect immunofluoresence, RT-PCR, indirect ELISA etc. was employed to detect expression and specificity of interest protein. Indirect immunofluorescence shows that cells with both separate transfection and co-transfection exhibit green fluorescence but noo fluorescence was observed by transfected null vector and un-transfected cells. RT-PCR shows interest gene was amplified in both transfected cells but corresponding interest product was not achieved in null vector transfected cells; Indirect ELISA shows cell supernatant of co-transfected recombinant plasmid can specifically combine with chick embryo allantoic fluid of IBV but not react with that of NDV, H5 and H9; there is no specific reaction between transfected null plasmid and un-transfected cell supernatant. It illustrates that we successfully express the specific antibody against IBV.Therefore, we developed a rapid method to amplify chicken’s antibody gene and further analysis of antibody gene massively amplified will facilitate the construction of chicken originated antibody database and selection of specific antibody gene. The construction of plasmid expressing gene heavy chain and light of IgY with histidine label, based on the full antibody gene amplified from spleens of chickens immunized with attenuated IBV vaccine, and their successful expression in 293T cells will lay a foundation for utilization of gene project to obtain the complete molecular study of specific antibody molecules, also, it will offer new direction for the subsequent massive production of specified monoclonal antibody and overcome the disadvantages of conventional methods, e.g., laber-consuming and expensive of those methods as well as issue of the mouse-originated molecules etc.