Preparation of Anti-Rabies Virus N Protein IgYs by DNA Immunization of Hens Using Different Types of Adjuvants

DNA immunization has been used to study vaccination methods and for production of specific antibodies. The present study aimed to apply DNA immunization to prepare specific IgYs, which react against rabies virus N protein (RV-N) and can be used to research and diagnose rabies virus. The DNA sequence of RV-N was ligated into a pcDNA 3.1 plasmid for constructing pcDNA-N. Eight hens were divided into four groups. Group 1 comprised the control group (non-immunized). In Groups 2, 3, and 4, hens were injected intramuscularly with pcDNA-N (400 µg/hen). Eight injections were administered every other week. From the 4th week, an adjuvant was injected in addition to pcDNA-N. Freund''s complete adjuvant (FCA) and λ-carrageenan were administered to Groups 3 and 4, respectively. Eggs were collected daily, and the specific antibody activities of egg yolks were measured by ELISA. IgYs were purified from pooled egg yolks at 16–19 weeks post-administration in each group. The detection sensitivities of the RV-N were compared using purified IgY as the primary antibody for ELISA, dot blotting, and western blotting. Egg yolks from one of the two hens in Group 2 (pcDNA-N alone) and all hens in Groups 3 (pcDNA-N + FCA) and 4 (pcDNA-N + λCarra) had increased ELISA values. The combined use of λ-carrageen in DNA immunization resulted in an adjuvant effect comparable to that of FCA. Each purified specific IgY detected RV-N in the ELISA, western blotting, and dot blotting; however, the detection sensitivity differed. Higher detection sensitivity of the +λCarra IgY was observed by ELISA, whereas there was higher detection sensitivity of +FCA IgY in western blotting and dot blotting. In summary, anti-rabies virus N protein IgY was prepared through DNA immunization of hens using FCA or λ-carrageenan as adjuvants and can be used as a primary antibody to detect rabies viruses.     

不同植物精油对体外瘤胃发酵及甲烷产量影响的比较研究

在底物精粗比为6∶4的条件下,在底物中添加不同剂量[使发酵液中植物精油的浓度分别为0(对照)、50、100、200和400 mg/L]的丁子香酚、D-柠烯、茴香脑、肉桂醛、百里香酚或香芹酚,通过体外产气法比较研究不同植物精油对体外瘤胃发酵和甲烷(CH4)产量的影响。每种植物精油的每个剂量设3个重复。体外模拟瘤胃发酵培养24 h,测定产气量和气体中的CH4含量以及发酵液的p H、挥发性脂肪酸(VFA)和氨态氮(NH3-N)浓度。结果表明:1)除百里香酚外,添加各种植物精油对体外发酵液p H均无显著影响(P0.05)。2)添加丁子香酚、D-柠烯、茴香脑和肉桂醛对体外发酵液总VFA浓度没有显著影响(P0.05),但总VFA浓度随百里香酚和香芹酚浓度的增加呈二次曲线变化(PQ0.01)。与对照组相比,添加400 mg/L百里香酚和香芹酚显著降低体外发酵液总VFA浓度(P0.01)。D-柠烯、茴香脑、百里香酚和香芹酚的添加改变了各VFA占总VFA的摩尔百分比。与对照组相比,添加50 mg/L D-柠烯和茴香脑使乙酸比例显著增加(P0.05),丙酸比例显著降低(P0.05);而添加400 mg/L D-柠烯和茴香脑则使乙酸比例显著下降(P0.05),丙酸和丁酸比例显著上升(P0.05)。百里香酚和香芹酚的添加对乙酸比例没有产生显著影响(P0.05),与对照组相比,400 mg/L百里香酚和香芹酚使丙酸比例显著下降(P0.05)。3)添加茴香脑、百里香酚和香芹酚显著影响体外发酵液NH3-N浓度(P0.05),与对照组相比,400 mg/L百里香酚和香芹酚显著降低NH3-N浓度(P0.05)。4)添加D-柠烯、茴香脑、肉桂醛对体外发酵24 h产气量没有显著影响(P0.05)。与对照组相比,各浓度的百里香酚和香芹酚均显著降低体外发酵24 h产气量(P0.05),且产气量随百里香酚和香芹酚浓度的增加呈二次曲线变化(PQ0.01)。5)添加D-柠烯、茴香脑和肉桂醛对体外发酵24 h CH4产量没有显著影响(P0.05)。与对照组相比,50和100 mg/L的丁子香酚显著增加体外发酵24 h CH4产量(P0.05),而400 mg/L的百里香酚和香芹酚体外发酵24 h CH4产量分别降低84.7%(P0.05)和73.9%(P0.05)。综合以上试验结果可知,不同植物精油对体外瘤胃发酵和CH4产量的影响结果不同,且与添加剂量有关。其中,低剂量的百里香酚和香芹酚促进体外瘤胃发酵,而高剂量的百里香酚和香芹酚抑制体外瘤胃发酵且显著降低24 h CH4产量。