Localization Of Cytokines In Abomasum Of Goats Immunized With Recombinant Galectins Of Haemonchus Contortus

The galectins and cysteine protease of Haemonchus contortus play important roles at host pathogen interactions.Based on the previous studies,we carried out the following researches:1.Synthesis of Digoxigenin-labeled probesGoat IFN-γ,IL-2,IL-4,IL-6,IL-1βand TNF-αspecific primers were designed based on appropriate sequences published in the GenBank database and the plasmids containing the genes of cytokines kept in our laboratory were used as templates for each amplification reaction to generate probes by standard PCR.All probes were corresponding to the predicted sizes confirmed by gel electrophoresis.The concentrations of Digoxigenin-labeled probes were respectively 0.82μg/μL,0.56μg/μL,0.72μg/μL,0.69μg/μL,0.91μg/μL and 0.78μg/μL determined by the UV method.Dot blot confirmed that a minimum of 0.001ng probe was visualized after the color reaction.And the specificity of probes was also confirmed by in situ hybridization to goat peripheral blood lymphocytes stimulated by Con A and RT-PCR.2.Localization of cytokines in abomasum of goats immunized with recombinant galectins of H.contortusIn this research,we used in situ hybridization method to localize cytokines in abomasum of goats immunized with rHco-GAL-m/f and analyzed the number of cytokine mRNA~+ cells in each group.The results showed that cytokine mRNAs were detected specifically in the sub-mucosa layer of abomasum of all immunized or challenged goats, which was characterized with blue reaction signals,not in the control(group A).IL-4 and IL-6 mRNA~+ cells were significant difference(P＜0.05)among B,C and D group animals. Goats in group C had higher IL-4 and IL-6 when compared to B and D group.IL-1β,IL-2 and TNF-αmRNA~+ cells were significant difference(P＜0.05)among B,C and D group animals.Goats in group D had higher IL-1β,IL-2 and TNF-αwhen compared to B and C group.On the other hand,IFN-γmRNA~+ cells weren't significant difference(P＞0.05) among B and C group.Goats in group D had higher IFN-γwhen compared to B and C group and were significant difference(P＜0.05).These results indicated that rHco-GAL-m/f were capable of inducing local immune responses in abomasums of goats.3.Detection of galectins at several life stages of H.contortusNative proteins were extracted from the L1,L2,L3 and female and male adult H. contortus,and the concentrations of proteins were detected with Bradford method.Western blot was used to analyze and test the expression of galectins at the above life stages of H. contortus.Native proteins probed with rabbit anti-recombinant galectins serum displayed a single band of approximate 33kDa at the adult stages of H.contortus.Galectins of male and female H.contortus were recognized by both of the first antibodies respectively.The image analysis results showed that the mean grey degree of galectin hybridized band in female H.contortus was 1.4 times than the male.4.Detection of cysteine protease(Hc58)at several life stages of H.contortusWestern blot was used to analyze and test the expression of Hc58 at the L1,L2,L3 and female and male adult H.contortus.Native proteins prepared in the third chapter probed with goat anti-recombinant Hc58 serum displayed a single band of approximate 29kDa at the adult stages of H.contortus.The results suggested that Hc58 was specific staged expression at the life cycle of H.contortus.The image analysis results showed that the mean grey degree of Hc58 hybridized band in female H.contortus was 1.62 times than the male.