抗IBDV独特型抗体杂交瘤细胞株的建立及其产物生物学特性测定

Establishment and Biological Properties of Hybridoma Cell lines Secreting Anti-IBDV Idiotypic Antibodies

用纯化的抗IBDVIgG免疫Balb c小鼠 ,取其脾细胞与SP2 0细胞在聚乙二醇 (PEG)作用下融合 ,ELISA法检测筛选 ,经有限稀释法克隆 3次 ,获得 2株 (5F4株 ,2B6 株 )分泌抗IBDV独特型抗体的杂交瘤细胞株 ,其能诱生Balb c小鼠产生ELISA抗体效价分别为 1∶12 80 0和 1∶2 5 60 0的含抗IBDV独特型抗体的腹水。用此独特型抗体与福氏完全佐剂和福氏不完全佐剂乳化制备成抗IBDV独特型抗体疫苗 ,免疫接种SPF鸡和普通京白公鸡 ,然后用IBDV强毒株 (SD株 ) 2 0 0 0ELD50 攻毒 ,SPF鸡免疫组 5 0只 ,有 5只发病、2只死亡 ;对照组 10只全部发病 ,8只死亡。普通京白鸡免疫组 3 0只 ,有 7只发病 ,1只死亡 ;对照组 10只全部发病 ,6只死亡。经X2 检验 ,SPF鸡X2 =3 4 15 ,普通鸡X2 =16 68,查X2 值表得X2(1 ) 0 0 1 =6 63 ,SPF鸡X2 和普通鸡X2 均大于X2(1 ) 0 0 1 (P <0 0 1) ,由此表明抗IBDV独特型抗体疫苗具有很好的免疫原性 ,对易感日龄的SPF鸡和普通鸡均具有极其明显的保护作用。

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 (2B 6 strain,5F 4 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 IgG 1 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 2B 6) and 1:12800 (for 5F 4). 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 ELD 50 after two immunizations. 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. X 2 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.