抗伴放线放线杆菌IgY的制备及抑菌试验研究

Preparation of IgY against Actinobacillus actinomycetemcomitans and growth suppression of actinobacillus actinomycetemcomitans and capnocytophaga gingivalis by specific IgY against Actinobacillus actinomycetemcomitans

目的:观察伴放线放线杆菌诱导母鸡产生特异性IgY抗体情况,以及其抑制伴放线放线杆菌(A.a)和牙龈二氧化碳噬纤维菌(C.g)生长效果。方法:应用免疫接种法、水稀释法、盐析法、液体培养抑菌法、以及ELISA法,诱导、提取和纯化IgY抗体,取一定量抗体与细菌共同培养,测定抑制伴放线放线杆菌和牙龈二氧化碳噬纤维菌生长效果。结果:两步硫酸铵盐析沉淀的IgY抗体纯度达85.6%～90.3%;抗原结合效价为1∶32000;抗伴放线放线杆菌IgY抗体与牙龈二氧化碳噬纤维菌交叉免疫反应的抗原结合效价为1∶8000;当抗伴放线放线杆菌IgY抗体浓度在5.0、1.0、0.1g/L时,细菌浓度在5×108CFU/L培养24h其抑菌率分别为31.60%(P=0.004)、10.24%(P=0.024)、-3.30%,培养72h其抑菌率分别为64.20%(P=0.004)、53.21%(P=0.002)、11.20%。细菌浓度在1×108CFU/L培养24h其抑菌率分别为35.71%(P=0.004)、30.95%(P=0.012)、11.11%,培养72h其抑菌率分别为65.11%(P=0.005)、54.04%(P=0.002)、16.17%;5.0g/L的抗伴放线放线杆菌IgY与1×108CFU/L牙龈二氧化碳噬纤维菌培养24h其抑菌率为41.61%(P=0.005),培养72h抑菌率为86.99%(P=0.014)。结论:伴放线放线杆菌能够诱导母鸡产生高效价的特异性IgY抗体,该抗体在一定的浓度内有抑制伴放线放线杆菌和牙龈二氧化碳噬纤维菌生长的作用;伴放线放线杆菌与牙龈二氧化碳噬纤维菌存在着共同抗原。

Objective： To prepare specific IgY production using Actinobacillus actinomycetemcomitans （ A. a ） immunizing hen, and then to investigate anti-Actinobacillus actinomycetemcomltans IgY inhibiting growth of A. a and Capnocytophaga gingivalis（ C. g）. Methods： Using immunization method, water-dilution method, twotep ammonium sulfate precipitation, inhibiting bacteria growth test in liquid anaerobic culture, and ELISA, IgY were induced, extracted, purified, and inhibiting growth of A. a and C. g by the IgY was roundly evaluated. Results：The IgY purity reached to 85.6% -90.3% through 550 g/L and 330 g/L ammo- nium sulfate precipitation, and efficacy value was 1 ： 32 000. The IgY efficacy value of anti- A. a was 1 ： 8 000 against C. g in cross-reactivity. When IgY concentrations of anti-A, a were in the 5.0,1.0,0.1 g/L and concentration ofA. a was in the 5 × 10g CFU/L, the suppression rate of A. a growth were 31.60% （ P = 0.004 ）, 10.24% （ P = 0.024 ）, - 3.30% respectively during 24 h culture and were 64.20% （ P = 0.004） ,53.21% （ P = 0.002 ）, 11.20% respectively in 72 h culture. When the concentration of A. a was in the 1 ×10g CFU/L, the suppression rate of A. a growth were 35.71% （ P = 0.004 ）, 30.95% （ P = 0.012 ）, 11.11% respectively during 24 h culture, and were 65.11% （P =0.005） ,54.04% （P =0.002） ,16.17% respectively during 72 h culture. When 5.0 g/L IgY of anti-A, a was cultured with 1 x l0s CFU/L C. g for 24 h, the suppression rate of C. g growth was 41.61% （P = 0. 005）, and for 72 h it was 86.99% （P = 0.014）. Conclusion：The hen is able to be induced to produce high efficacy val e IgY of anti-A, a by A. a. The specific IgY of anti-A, a is capable of inhibiting A. a and C. g growth. There are common antigens and cross immunizing reactivity between A. a and C. g.