Zhu Rui-Liang, Cui Zhi-Zhong, Zhao Jing
Animal Science and Veterinary College, Yangzhou University, Yangzhou 225009, China.
Sheng Wu Gong Cheng Xue Bao. 2003 Jul;19(4):462-6.
In recent years, the prevention and cure of infectious bursal disease (IBD) have become more and more difficult due to the emergence of very virulent strains of infectious bursal disease virus (vvIBDV) and the variant strains of IBDV. In this research, the hybridoma cell lines which secretes anti-idiotypic antibodies against anti-IBDV IgG were established. According to the Jerne's theory of immune network, the use of the anti-idiotypic antibodies as a vaccine will be a new method for the prevention of IBD. In this study, the SPF chickens were inoculated with the IBDV- SD strain, and the bursal was obtained from the died chickens. The bursal was then homogenized and frozen-thawed 3 cycles, and the virus samples were prepared by cane sugar density gradient centrifugation and dialysis. Typical IBDV particles were observed under an electron microscope, and the concentration of the virus protein measured by ultraviolet absorbance spectrophotometry was 10.8 mg/mL. SPF chickens were immunized with the virus and the highly immunized sera were prepared and purified by Sulfuric acid ammonia salt out and Sephadex G-25 chromatography. Then, Balb/C mice of six or eight weeks old were immunized interapertoneally(I. P.) with purified antibodies to IBDV at regular intervals. SP2/0 myeloma cells were fused with the spleencytes from the immunized mice at a ratio of 10:1, in 50% polyethylene glycol (1540) and were then cultured in HAT until all the SP2/0 cells died. The hybridoma cells were selected by ELISA and the highly positive holes were cloned 3 times with the method of limited dilution. Two strains (2B6 strain,5F4 strain) of hybridoma cells were obtained, which were shown by ELISA to steadily secrete anti-IBDV idiotypic antibodies. The chromosome number of the two hybridoma cells were about 88 - 106, 95 in average, and the antibodies secreted belonged to the types of IgG1 and Kappa. Balb/c mice of 3 months old were inoculated I.P. with about 10(7) hybridoma cells per capita, and the ascites were collected 12 days later and the titre of anti-IBDV idiotypic antibodies measured by ELISA was 1 :25600 (for 2B6) and 1:12800 (for 5F4) . The ascites containing the anti-IBDV idiotypic antibodies were emulsified with complete or incomplete Freund's adjuvants, and the anti-IBDV idiotypic antibody vaccine was obtained. SPF and common Jingbai chickens were immunized with the vaccine obtained. The immunized chickens with the vaccine were inoculated with IBDV-SD strain at a dose of 2000 ELD50 after twoimmunizations. All the 10 SPF chickens in the non-immunized group were sick, and 8 of them died; and 5 out of the 50 SPF chickens immunized group got sick and 2 died. All the 10 common Jingbai chickens in the control group were sick, and 6 died; 7 of the 30 immunized common Jingbai chickens got sick and only 1 died. Chi2 analysis showed that the difference between the immunized and the non-immunized groups in both the SPF and the common Jingbai chickens were significant (P < 0.01). Our result indicated that the anti-IBDV idiotypic antibody vaccine well protected chickens and had a great potential in both research and clinical application.
近年来，由于超强毒力传染性法氏囊病病毒（vvIBDV）和传染性法氏囊病病毒变异株的出现，传染性法氏囊病（IBD）的防治变得越来越困难。本研究建立了分泌抗IBDV IgG抗独特型抗体的杂交瘤细胞系。根据Jerne的免疫网络理论，使用抗独特型抗体作为疫苗将是预防IBD的一种新方法。在本研究中，用IBDV-SD株接种SPF鸡，从死亡鸡中获取法氏囊。然后将法氏囊匀浆并冻融3个循环，通过蔗糖密度梯度离心和透析制备病毒样本。在电子显微镜下观察到典型的IBDV颗粒，用紫外吸收分光光度法测得病毒蛋白浓度为10.8mg/mL。用该病毒免疫SPF鸡，制备高免血清，并通过硫酸铵盐析和Sephadex G-25柱层析进行纯化。然后，每隔一定时间给6周龄或8周龄的Balb/C小鼠腹腔注射纯化的抗IBDV抗体。将SP2/0骨髓瘤细胞与免疫小鼠的脾细胞按10:1的比例在50%聚乙二醇（1540）中融合，然后在HAT培养基中培养，直到所有SP2/0细胞死亡。通过ELISA筛选杂交瘤细胞，并用有限稀释法对高阳性孔进行3次克隆。获得了两株杂交瘤细胞株（2B6株、5F4株），ELISA结果显示它们能稳定分泌抗IBDV独特型抗体。两株杂交瘤细胞的染色体数约为88 - 106条，平均为95条，分泌的抗体属于IgG1和κ型。给3月龄的Balb/c小鼠腹腔注射约10⁷个杂交瘤细胞/只，12天后收集腹水，用ELISA检测抗IBDV独特型抗体的效价为1:25600（2B6株）和1:12800（5F4株）。将含有抗IBDV独特型抗体的腹水与完全或不完全弗氏佐剂乳化，得到抗IBDV独特型抗体疫苗。用所得疫苗免疫SPF鸡和普通京白鸡。免疫两次后，给免疫疫苗的鸡接种2000 ELD50剂量的IBDV-SD株。未免疫组的10只SPF鸡全部发病，其中8只死亡；免疫组的50只SPF鸡中有5只发病，2只死亡。对照组的10只普通京白鸡全部发病，6只死亡；免疫组的30只普通京白鸡中有7只发病，仅1只死亡。χ²分析表明，SPF鸡和普通京白鸡免疫组与未免疫组之间的差异均具有显著性（P < 0.01）。我们的结果表明，抗IBDV独特型抗体疫苗对鸡具有良好的保护作用，在研究和临床应用中都具有很大的潜力。
