Screeing And Active Detection Of Nanobody For The Vp1 Protein Of Avian Encephalomyelitis Virus

Avian encephalomyelitis virus(AEV) mainly invades the central nervous system of young chicks, which is mainly characteristiced by ataxia, tremors and paralysis of chicken, quail and turkey. Laying hens infected with AEV, which is no obvious neurological symptoms, but their egg production temporarily appear to decline. The VP1 protein of avian encephalomyelitis virus is a major host-protective immunogen, which is highly conserved, containing most of the specific neutralizing sites and major neutralizing epitopes. Currently, AEV is shorted of effective diagnostic and treatment method can only be prevented through immunization. So, the development of efficient, specific and inexpensive antibody for AEV is exigented, which will be more conducive to the early detection and research of AEV. Nanobody(Nb or VHH) with antigen affinity and specificity, stability, low cost, and ease of mass production, which will be possessed of broad prospects in pathogen detection and research. In our study, AEV VP1 protein as a bait protein, which was used to screen nonimmune Bactrian camel Nanobody yeast library by yeast two-hybrid system to obtain nanobodies with better active. This can be used for antigen detection and research on etiology of avian encephalomyelitis virus. Our work obtained following results.1. The VP1 gene was amplified from the plasmid of PMD-18 T of AEV ShaanXi strain, which was used the method of polymerase chain reaction with a pair of specific primers containing double enzyme site of EcoR I、Sal I, named pBD-VP1. The result of sequence alignment for pBD-VP1 showed that the cloned gene sequence of VP1 was not mutated. The result of toxicity and autoactivation to pBD-VP1 showed that was not toxicity and antoactive, which can be used for secrening Bactrian camel nanobody yeast library.2. 4 ml bait bacteria [Y2H Gold(pBD-VP1)]with 30℃ overnight cultured and 1 ml library bacteria [Y187(pAD-VHHs)] were mated. Hybridized combination after certificating was applied to 50 150 mm DDO/Aba/X-α-gal plate with cultured for 3 d~5 d at 30 ℃; finaly, 200 positive blue colones on the DDO/Aba/X-α-gal plate were obtained and marked. In order to reduce false positives, randomly selected 40 preponderant blue colones from the 200 colones were secondly spreaded on the QDO/Aba/X-α-gal to cultivate and obtained 9 more positive coloned. 9 colones respectively were co-transformated into Y2 HGold yeast strain to validate. Sequenced and comparative analysis for 9 colones; evently, 8 stains of anti-AEV SX VP1 proten antibodies were obtained.3. The recombinant plasmid pET28as-VHH was transformed into BL21(DE3) competent cells to optimize the expressional conditions of Inductive temperature, time and Concentration of IPTG. the results showed that finial optimal Inductive temperature was 30 ℃, optimal Inductive IPTG Concentration was 5 mmol/L, optimal Inductive time was 3 h. The purified production was analysised by DS-PAGE assay. The results showed that the weight of 8 stains of anti-AEV SX VP1 protein recombinant antibodies were approximately 34 ku, the results in line with expectations.4. The active in action was detected by indirect ELISA. The results showed that average OD450 value of Undiluted positive control wells was 1.056 [OD450=1.056(OD450>1)], and that average OD450 value of Undiluted negative control wells was 0.048[OD450=0.048(OD450 ﹤ 0.1)]. The positive and negative controls have been set up through reference criteria provided by IDEXX Company. The OD450 value of labeling protein as second negative control was less than 0.05; this indicated that the response was very weak. The reactogenicities of VHH4 and VHH9 were higher than the positive control; also VHH9 reactogenicity was higher than VHH4, the reactogenicities of other remaining six strains less than the positive control.5. The active in action of was detected by neutralization test in chicken embryo.the results showed that neutralization titer of positive serum was 1:717, neutralization titers of VHH9 and VHH4 respectivly were 1:609 or 1:502. It showed that VHH9 and VHH4 had better neutralization titer against AEV.In conclusion, Non-immune Bactrian camel Nanobody yeast library was screened by yeast two-hybrid system. 8 strains of anti-AEV SX VP1 protein recombinant antibodies were successfully obtained by these methods of co-transformated into Y2 HGold yeast strain to validate, Sequenced and comparative analysis. 8 strains of purified anti-AEV SX VP1 protein recombinant antibodies were obtained and the reactogenicities of these antibodies were detected by indirect ELISA and neutralization test in chicken embryo. Eventually, obtained two strains of antibodies with better activity in indirect ELISA laid a foundation for the antigen detection and etiological research of AEV.