对鸡卵黄免疫球蛋白两种提取方法的比较

本文比较了水稀释-饱和硫酸铵沉淀法（法一）和水稀释-冰乙醇分级沉淀法（法二）分离提纯鸡卵黄免疫球蛋白（IgY）的产率、纯度及抗体效价。结果表明：两种方法提取的IgY纯度均较高,法一纯化的IgY的产率及蛋白活性相对更好。     

抗CP型、NCP型牛病毒性腹泻病毒高免卵黄抗体的制备

本研究采用致细胞病变（cytopathic,CP）型牛病毒性腹泻病毒（bovine viral diarrhea virus,BVDV）标准毒和非致细胞病变（noncytopathic,NCP）型新疆优势毒株免疫产蛋鸡,用改良PEG法提取卵黄抗体(IgY),并对提取的IgY采用SDS-PAGE检测纯度,间接ELISA检测免疫后每隔7 d的抗体效价,并测定所得抗体对NCP型BVDV的中和效价。结果表明,用该法提取的IgY纯度较高;间接ELISA结果证明,经过4次免疫后,抗CP型BVDV的效价达到1∶32000,抗NCP型BVDV的效价达到1∶40000,3个月后再次检测,卵黄抗体效价未见明显下降。最后一次免疫14 d的抗体对NCP型BVDV的中和效价达到1×10_^-3。     

Egg yolk IgY against RHDV capsid protein VP60 promotes rabbit defense against RHDV infection

VP60 capsid protein is the major structural and immunogenicity protein of RHDV (Rabbit hemorrhagic disease virus, RHDV), and has been implicated as a main protein antigen in RHDV diagnosis and vaccine design. In this report, egg yolk antibody (IgY) against N-terminal of VP60 was evaluated and developed as a new strategy for RHDV therapy. Briefly, N-terminal of VP60 (～250aa) fragment was cloned and inserted into pET28a expression vector, and then the resultant plasmid, pET28a/VP60-N, was transformed into E. coli BL21(DE3) for recombinant VP60-N protein (rVP60-N) expression. Next, the rVP60-N was purified by Ni⁺-affinity purification chromatography and identified by Western blotting with RHDV antiserum. After immunizing the chickens with rVP60-N, the anti-rVP60-N IgY was isolated, and the activity and specificity of the IgY antibody were analyzed by ELISA and Western blotting. In our results, the rVP60-N could be expressed in E. coli as soluble fraction, and the isolated anti-rVP60-N IgY demonstrated a high specificity and titer (1:22,000) against rVP60-N antigen. For further evaluation of the IgY efficacy in vivo, rabbits were grouped randomly and challenged with RHDV, and the results showed that anti-rVP60-N IgY could significantly protect rabbits from virus infection and promote the host survival after a sustained treatment with anti-rVP60-N IgY for 5 days. Taken together, our study demonstrates evidence that production of IgY against VP60 could be as a novel strategy for the RHDV therapy.     

Immunostimulatory effects of fermented vegetable product on the non-specific immunity of Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

The stimulatory effect of fermented vegetable product (FVP) upon the phagocytic and superoxide generation of leukocytes was studied in the Japanese flounder Paralichthys olivaceus. The phagocytic activity of casein-induced, intraperitoneal leukocytes was investigated and quantified, that is the activity significantly increased (P<0.05 or <0.01) by the addition of FVP beyond 3 mg/kg body weight. Further analysis investigated the effect of FVP on superoxide generation in leukocytes. Established in vitro cytochrome c reduction assay was used to measure superoxide generation; reduced levels of FVP in assay samples had a profound effect on superoxide generation. FVP was also incorporated in commercial diets and fed to Japanese flounder for 4 weeks. The phagocytic activities and superoxide generation of peritoneal induced leukocytes were significantly higher (P<0.05, <0.01) in fish fed the FVP supplemented diet than fish fed the control diet. FVP feeding in fish had a significantly higher (P<0.05) activity of lysozyme than in the control fish.     

Effect of farming patterns on growth,antioxidant activity and nonspecific immunity of loach,Paramisgurnus dabryanus

This study was carried out to compare growth, intestinal enzymes activity, antioxidation and nonspecific immunity of loach between farmed and imitative ecological farming loach, Misgurnus anguillicaudatus. Loach from farmed (average body weight 15.03 ± 0.36 g) and imitative ecological farming (average body weight 12.44 ± 0.62 g) population were used to analyse growth, whole-body composition, intestinal enzymes, antioxidant enzymes activity and resistance to bacteria. The results showed specific growth rate, average daily gain and protease, lipase, amylase and γ-GT activities in intestine and hepatopancreas of farmed loach were significantly higher (p < 0.05) than of imitative ecological farming loach. However, total protein, lysozyme and albumin contents and superoxide dismutase, catalase and glutathione peroxidase activities of imitative ecological farming loach were significantly higher (p < 0.05) than of farmed loach. Moreover, resistance to Aeromonas hydrophila of imitative ecological farming loach was higher than of farmed loach (p < 0.05). Taken together, ecological farming loach possesses higher antioxidant capacity and resistance to bacteria than farmed loach.     

精制禽流感高免卵黄抗体的研究

用禽流感油乳剂灭活苗按一定的免疫程序免疫SPF蛋鸡,制备高免卵黄抗体,用改良的水稀释法对IgY进行纯化,经过抗体效价检测、无菌检验、安全试验、动物试验,该卵黄抗体可以用于田间试验。保存期试验证明:精制禽流感高免卵黄抗体室温条件下可保存10个月,4℃条件下保存15个月,-20℃条件下保存18个月。     

抗轮状病毒IgY的研制

用鸡卵黄制备抗轮病毒（RV）免疫球蛋白，预测和治疗婴幼儿RV感染，制备RV抗原，通过不同途径免疫母鸡，几种纯化方法联合应用从鸡卵中提取特异性鸡卵黄抗体（IgY),进行原理化学检定及动物效力实验，结论：已成功地应用RV免疫母鸡制备出高效价特异性抗RV－IgV.     

抗ETEC F18+菌毛特异性IgY的稳定性研究

用间接ELESA方法测定了抗F18^＋ETEC IgY对温度、酸碱度、反复冻融、渗透压、胃蛋白酶、胰蛋白酶的稳定性，并考察非还原性糖（乳糖、蔗糖）对IgY活性的影响。结果表明：60℃水浴30min效价无变化；pH4．0～11．0环境中，IgY活性改变不显著；经7次反复冻融和50％蔗糖渗透压处理，抗原结合活性也几乎未受损失；pH2．5时，该IgY对胃蛋白酶非常敏感，经胃蛋白酶37℃处理4h，效价从1：7200下降到1：450；37℃时分别经胃蛋白酶（pH4）和胰蛋白酶（pH7，5）各处理4h，效价均无变化；在胃蛋白酶的处理中，蔗糖和乳糖对该IgY具有一定的保护作用。