| Ticks represent a group of highly specialized bloodsucking arthropods that are obligate temporary parasites of mammals,birds,and reptiles.They are well known vectors not only transmitting a great variety of infectious pathogens,but also having a super reproductive ability,and causing serious damage to human and domestic animals.At present,the reproductive physiology of ticks has become one of the international focus research realms.Many modern biological technologies and the methods of the cross correlative realm, such as biological chemistry,immunocyte chemistry,ELISA and GC/MS,were applied in these researches.Based on the purification of the vitellin(Vn),monoclonal antibody(McAb) against Vn of the hard tick Haemaphysalis longicornis,which was distributed widely in China,was produced for the first time and a series of identifications were done about the McAb.Based on these work,vitellogenin(Vg) or Vn contents of fat body,hemolymph and ovary were determined using double antibody sandwich ELISA,Vg was purified and properties were investigated.The present research could be of important scientific significance for elucidating the vitellogenesis and regulation mechanism and revealing the reproductive biological characteristics,the adaptive strategy and the physiological regulation in ticks.The main results were as follows:1.Six hybridoma cell lines secreting monoclonal antibody against Vn of the hard tick 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 1B5,2A7,2B8, 2F2,3A1 and 3G1,respectively.2.The isotypes of McAbs were identified using mouse monoclonal antibody isotyping reagents of the Sigma company.The results indicated that 1B5,2B8 and 2F2 were of the isotype IgGA,2A7 was of the isotype IgGl,while 3A1 and 3G1 were of the isotype IgG2a.3.All the six antibodies had high specificity and affinity,and their titers were over 10-5 using double antibody sandwich ELISA and indirect ELISA.4.SDS-PAGE and affinity analysis of 1B5,whose specificity and titer were the highest, indicated that the molecular masses of its heavy chain and light chain were 58kD and 21kD, respectively,and the affinity constant was 2.8993×10-6.The affinity of 1B5 was the best.5.Western blot analysis showed that the six antibodies had specific immunological reaction with the eight subunits of the Vn.6.The Vg or Vn contents of the central and peripheral fat body,hemolymph and ovary were determined using double antibody sandwich ELISA.The result showed that the central and peripheral fat body and the hemolymph were beginning to produce Vg after two days of engorgement,but the content of the hemolymph was fewer.The Vn of ovary was appeared after four days of engorgement.The above results revealed that the Vg was synthesized at the midgut and fat body,released to hemolymoph and then took it selectively by growing oocyte to produced Vn,which as the resource of fetation,in the vitellogenesis of H.longicornis.7.The Vg of H.longicornis was a glycolipoprotein,which was only one kind of protein and the molecular weight was 487 kD,composed of seven protein subunits.The molecular weight of the seven protein subunits were 165 kD,134 kD,112 kD,80 kD,78 kD,71 kD and 65 kD.8.The analysis of Vg 2-DE showed that 16 protein spots were found in Vg.The protein spots of P4 and P6 were single proteins each other and their pI were between 5 and 8.9.Vg lipids were detected by GC/MS.The results show that lipids were mainly hexadecanoic acid and octadecanoic acid.
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