Preparation Of Antibodies To The Vitellin And Studies On The Vitellogenesis Of Parthenogenesis Population Of Haemaphysalis Longicornis (Acari: Ixodidae)

Many modern biological technologies, such as biological chemistry and immunocyte chemistry, were applied in these researches. Based on the purification of the vitellin (Vn), polyclonal antibody (PcAb) and monoclonal antibody (McAb) against Vn of parthenogenesis population of Haemaphysalis longicornis were produced for the first time and a series of identifications were done about the McAb. Vitellogenin (Vg) or Vn contents of perivisceral fat body, parietal fat body, hemolymph and ovary were determined using double antibody sandwich ELISA. The present research could be of important scientific significance for elucidating the vitellogenesis. The main results were as follows:1. The polyclonal antibody against Vn of parthenogenesis population of H. longicornis were produced by serum from rabbit which were immunized with the Vn. The titers of PcAb was over 1:768000 using indirect ELISA.2. Seven hybridoma cell lines secreting monoclonal antibody against Vn of parthenogenesis population of H. longicornis were produced by fusing myeloma cell (SP2/0) with spleen cell, both from BALB/c mouse which were immunized with the Vn. They were named as 1F6,2H6,3A9,4G11,4H1,5G11 and 6G2.3. The titers of 1F6,4H1 and 6G2 were over 1:819200,and the titers of 2H6,3A9,4G11 and 5G11 were over 1:1638400 using indirect ELISA.4. The isotypes of McAbs were identified using mouse monoclonal antibody isotyping reagents of the Sigma company. The results indicated that 1F6 and 3A9 were of the isotype IgG2a, 2H6, 4G11, 5G11 and 6G2 were of the isotype IgG2b, while 4H1 was of the isotype IgG1.5. All the seven antibodies had high specificity and affinity using double antibody sandwich ELISA.6. The 4G11 had the highest specificity and titer. The titer of the purified 4G11 was over 1:819200 and the affinity constant was 4.6×10~7.7. SDS-PAGE analysis of 4G11, which was purified, indicated that the molecular masses of its heavy chain and light chain were 55.8KD and 20.9KD respectively. 8. The purified 4G11 had specific immunological reaction with only two subunits of the Vn using western blot analysis.9. The Vg or Vn contents of the perivisceral and parietal fat body, hemolymph and ovary were determined using double antibody sandwich ELISA. The result showed that the Vg was appeared in hemolymph at the day of engorgement. The perivisceral and parietal fat body were also beginning to produce Vg at the day of engorgement, but the content was fewer. The Vn of ovary was appeared after four days of engorgement. The above results revealed that the Vg was synthesized at the fat body. Once synthesized, Vg quick release to the hemolymoph. And then took it selectively by growing oocyte to produced Vn, which as the resource of fetation, in the vitellogenesis of parthenogenesis population of H. longicornis.