| H9N2 subtype avian influenza(AI)is a type of low pathogenic avian influenza.This disease mainly infects poultry and wild birds and is widely prevalent in many parts of the world,causing huge economic losses to the poultry industry.The clinical symptoms of H9N2 include respiratory distress,decreased egg production,and susceptibility to secondary infection by other pathogens.Hemagglutinin(HA),a glycoprotein on the surface of avian influenza virus,is the main protective antigen of AIV,eliciting the production of neutralizing antibodies and providing protection to the host.In this study,the recombinant protein of H9N2 subtype avian influenza virus HA expressed by rice endosperm expression system was purified and immunized in BALB/C mice.Preliminary evaluation of the immunization effect of the rice-derived HA protein was performed to lay the foundation for the study of H9N2 subunit vaccine.Meanwhile,immunochromatographic test strips for the detection of antibodies to H9N2 subtype avian influenza virus were prepared in this experiment to provide technical support for the detection of antibodies to H9N2 avian influenza.The results of this study were as follows.1.Purification of rice-derived HA protein of H9N2 subtype avian influenza virusRice seeds containing HA protein were ground into powder with a grinder,and the protein was extracted by adding 5 times the volume of extraction buffer(50 mmol/L Tris 10 mmol/L NaCl pH 9.0)to obtain the crude extract of HA protein.Then HA protein was enriched by Q anion exchange chromatography,and the results showed that HA protein was enriched at 25%elution condition.Then the 7E10G8 monoclonal antibody was purified by HiTrapTM Protein A HP antibody purification column to prepare an HA-specific antibody affinity chromatography column,which was used to further purify the HA protein.Subsequently,the purified HA protein was detected by SDS-PAGE and Western-blot.The results showed that the purity of HA protein reached more than 90%,and the HA protein strip gel on SDS-PAGE was sent to Biotech Bioengineering(Shanghai)Co.The results showed that the amino acid sequence of the protein was consistent with that of HA protein of H9N2 subtype avian influenza virus,and the molecular weight of the recombinant protein was about 130 kDa.2.Preliminary evaluation of the immunization effect of rice-derived HA protein of H9N2 subtype avian influenza virusThe purified recombinant HA protein was mixed with adjuvant and immunized by subcutaneous injection into BALB/C mice to initially evaluate the immunization effect of HA protein.Six different HA protein immunization dose groups were set up,namely 0.5μg,1μg,3μg.6μg and 9μg,along with positive control groups(commercial inactivated vaccine)and negative control groups.As a result,among the five experimental groups,the 3 μg,dose group had the highest antibody potency of 7.5log2 at 3 weeks after secondary immunization,and the other experimental groups had the highest to lowest antibody potency,in the order of 1 μg,6μg,9 μg and 0.5 μg.This result indicates that antibody potency increases with increasing dose(e.g.,0.5 μg to 3 μg);However,when the immunization dose increases to a certain level,the potency decreases with increasing protein dose(e.g.,3 μg to 9 μg),demonstrating that more immunogen is not more effective.The results of the positive experimental group showed that the HI potency reached 4.25 log2 at day 7 after the first immunization and 7.5 log2 at week 3 after the second immunization,which was consistent with the experimental group(3 μg dose group),while the antibody potency of the negative control group did not increase throughout the experiment.The results of the serum neutralization test showed that the neutralization potency of both the experimental group(3 μg dose group)and the positive control group reached 8 log2 before challenge;while the HI titer of the other dose groups of the experimental groups ranged from 6 log2 to 8 log2.The mice were then attacked at a dose of 106 EID50/0.1 mL at week 3 after the second immunization,and the clinical symptoms were observed.The positive control group and the experimental group both showed normal performance.However,the negative group showed symptoms such as weight loss,decreased feed intake,hairy hair and bad mental state.And the results of the post-attack body weight measurement showed that the 3 μg dose group and the positive control group lost less weight and recovered faster than the other groups.In order to detect the amount of virus titer carried by the mice after the attack,the lungs of the mice in each groups were collected on the 4th day after challenge.The results showed that the virus titers were lowest in the 3 μg dose group and positive control group(about 2 log10)and highest in the negative control group(about 6.6 log10),with highly significant differences between the 3 μg dose group and the negative control group(P<0.01).Taken together,the results suggest that the H9N2 HA protein expressed in rice lays the foundation for the development of a novel H9N2 subtype avian influenza subunit vaccine.3.Preparation of colloidal gold immunochromatographic test strip for the detection of antibodies to H9N2 subtype influenza virus.In this study,colloidal gold-labeled purified rice-derived HA protein was used and immobilized on a binding pad.The test strips were prepared by spraying rabbit anti-chicken multiple anti-IgG at a concentration of 2.34 mg/mL on a nitrocellulose membrane as the test line(Test line)and H9N2 subtype AIV HA mouse monoclonal antibody at a concentration of 2 mg/mL on the membrane as the quality control line(Control line).The performance of the test strips was tested.The results showed that the test paper reacted well with H9N2 standard positive sera and did not react with H5N1 and Newcastle disease standard positive sera,indicating that the test paper had strong specificity for H9N2 antibody and no cross-reactivity with other virus sera;and the test paper had high compliance with HI test results;Meanwhile,the H9N2 antibody test paper had high sensitivity and higher sensitivity than HI results(about 9.8 times).In conclusion,we have established a preliminary method for the detection of antibodies to avian influenza N9N2 subtype,and this method is convenient,rapid,highly sensitive and specific. |
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