Preparation Of Immune Camel Milk And Its Protective Effect On Mice Infected With Salmonella Typhimurium

Camel is important economic livestock in arid or semi-arid regions.The camel continues to be vital to daily life as a source of milk and meat,and a means of transportation.Immunoglobulin G(IgG)in Camelids differs from all other known antibodies,which consists conventional IgG1 and heavy-chain antibodies(IgG2 and IGG3).Compared with conventional IgG,HCAbs have smaller molecular weight,higher stability,stronger affinity and solubility,and strong penetration,so it has been widely used.In order to make full use of Bactrian camels,we prepared anti-diarrhea immune camel milk and studied their specific HCAbs systematically,which might provide a scientific basis for its application in health foods and functional foods.The main results were as follows:1.Preparation of anti-diarrhea immune camel milk and the determination of the antigen-binding activity of its specific IgG subclassesIn this chapter,we first reported the preparation of immune camel milk from immunization of lactating camels with a multivalent vaccine consisting of 3 strains of pathogenic diarrhea bacteria.We used Protein A/G chromatography affinity to purify each IgG subclass followed by SDS-PAGE under reducing and non-reducing conditions to detect the purifications,and then determined the concentration by BCA kit.We established indirect Enzyme-linked immunosorbent assay(Elisa)protocol to detect the antigen-binding activity of each IgG subclass in the serum and whey.The results as follows:(1)The relative titers of immune serum(1:5000)and whey(1:1000)were significant increased after the immunization.It was indicated that specific antibodies were appeared in serum and whey of camels in immune group.(2)The reducing SDS-PAGE results revealed that the purified total IgG was composed of three heavy-chains with molecular weight as 40 kDa,45kDa and 55 kDa,and one lightchain with molecular weight of 27 kDa,which upon non-reduction yielded two molecules with 180 kDa and 110 kDa,respectively.The purified IgG1 has a molecular weight of ~180kDa(non-reduced)and dissociated upon reduction into one heavy chains of 55 kDa and one light chains of 27 kDa.Two band of heavy chains with molecular weight of around 40-50 kDa were detected from purified IgG2 under reduced condition,and with molecular weight of 110 kDa under non-reduced condition.(3)The content of each IgG in the immunized serum was significant higher than control group(p<0.05),while the content of each IgG in immunized whey was increased slightly,but there was no significant difference with the control group(p>0.05).(4)The results of indirect Elisa showed that immunized camels produced both conventional and HCAbs specific to the antigen.The titers of each IgG subclass in the immune whey and serum was ranged from 1:4000 to 1:64000,which was 2 to 64 times higher pre-immune.Our findings indicated that immunization of lactating camels can produce immune milk containing specific IgGs,which may be exploited in therapies for prevention of pathogenic-induced diarrhea.2.The antigen-binding ability of each purified IgG subclasses from anti-diarrhea immune camel whey and serum under variation conditionsIn this chapter,we first reported the antigen-binding residual ability of each purified IgG subclass under different temperature,pH,and protease hydrolysis conditions by indirect ELISA assay.Moreover,we studied the protective effect of different sugar protectant on the antigen-binding ability of each IgG subclass under heat treatment and low pH conditions.The result as follows:(1)The thermal stability of HCAbs was significantly higher than that of IgG1 under65?~100 ?(p<0.05).The antigen-binding ability of each IgG subclass followed second order reaction.The thermodynamic parameters of each IgG subclass was different.(2)The total IgG and IgG1 was stable under neutral or alkaline conditions.The HCAbs with higher stability between pH4.0~pH10.0.The loss rate of each IgG antigen-binding losses at pH>10.0 was much lower than that at pH<4.0.(3)The protective effect of maltose,mannitol,and glycine on each purified IgG subclass was different under heating and lower pH conditions,and the optimal protective agent for each IgG subclass was also different.(4)In artificial gastric juice(pH2.0 and pH4.0),the most important influence factor of the antigen-binding ability of each IgG subclass was pH value,followed by the amount of pepsin.In the artificial intestinal juice,the antigen-binding ability of each IgG subclass was mainly influenced by the amount of trypsin,followed by digestion time.3.In vitro antibacterial effect of immunized and non-immunized camel whey IgGs and its protective effect on Salmonella typhimurium(ST)-infected mice(1)In vitro antibacterial experiments showed that both immunized and non-immunized IgGs inhibited the growth of ST,and the inhibitory effect of immunized IgGs on ST was significantly higher than that of non-immunized camel whey IgGs(p<0.05).The TEM results showed that the immunized camel whey IgG could separate the cytoplasmic wall of the bacteria cells,and the structure was destroyed.The non-immunized IgG subtypes had no obvious effect on the cell structure of the ST.(2)In vivo protective effect on ST-infected mice showed that IC and NIC group could effectively relieve the body weight loss of mice caused by ST infection,and significantly reduced the number of ST colonies in feces(p<0.05).Both of them could significantly reduce the ST colonies in liver and spleen(p<0.05).The levels of TNF-??IL-1??IL-6 and IFN-? was significantly decreased in serum,colon and ileum,and the anti-inflammatory cytokines of IL-10 and TGF-1? was significantly increased than that of ST group(p<0.05).Both of them significantly increased the expression of MUC2 and LYS in the colon and ileum of ST-infected mice(p<0.05).In addition,both of them could significantly increase the expression of TNF-??IL-1??IL-6 and IFN-? in liver and spleen,and could down-regulate the expression of IL-10 and TGF-1? in ST-infected mice(p<0.05)?The results showed that both IC and NIC could relieve systemic inflammation caused by ST and provided a protective effect on the intestinal barrier,and the IC group showed better effect than that of NIC group.