摘要
目的评价硫糖铝对HpaA IgY的保护作用,为制备IgY口服制剂奠定基础。方法大量诱导工程菌DH5α-HpaA-pQE30,制备纯化的重组蛋白HpaA。以HpaA为抗原接种洛曼母鸡,制备纯化的IgY。在不同pH值、含不同浓度胃蛋白酶的HpaA IgY液中加入不同浓度的硫糖铝;将IgY液反复冻融;以ELISA法测定HpaA IgY的剩余抗体活性(AR/AC)。结果在pH 1.5条件下,60%硫糖铝可使IgY抗体剩余活性维持65.8%;在pH 2.0条件下,含30%硫糖铝的IgY可使抗体活性维持85.4%,含50%硫糖铝的IgY可完全维持抗体活性。在pH 3.0条件下,含30%硫糖铝的IgY可使抗体活性维持87.3%,而含40%以上硫糖铝的IgY几乎可完全维持抗体活性。在胃蛋白酶浓度0.02mg/mL条件下,当含10%、30%、50%硫糖铝IgY的剩余活性,在pH值2.0时,分别为62.8%、71.6%、82.5%;在pH值3.0时,分别为75.6%、87.4%、94.5%;反复冻融后,含10%硫糖铝的IgY可使抗体活性提高到70.6%,含30%硫糖铝可使IgY的抗体活性提高到82.7%,50%硫糖铝可提高到89.3%。结论 30%以上的硫糖铝可增强VacA IgY对低pH和胃蛋白酶的耐受能力,抗冻融能力,是较理想的IgY保护剂。
Objective To evaluate the protective effects of sucralfate on anti-H.pylori HpaA immunoglobulin yolk（HpaA IgY） and provide experimental basis for preparation of IgY by oral administration.Method The recombinant protein HpaA was expressed in engineering bacteria DH5α-HpaA-pQE30 and purified.HpaA IgY was prepared by inoculating purified HpaA in Lohman hen and then depurated.Sucralfate of different concentration was added into HpaA IgY under different pH（1.0-3.0） and contained different concentration of pepsin,and was enforced with freeze thawing.The residuary antibody activity（AR/AC%）of IgY was determined by the method of ELISA.Results The activity of HpaA IgY containing 60% sucralfate could keep its activity at 65.8% under pH 1.5.The activity of IgY containing 30% sucralfate could keep its activity at 85.4% while 50% sucralfate could completely keep its activity under pH 2.0.Under pH 3.0,30% sucralfate could remain its activity at 87.3% while sucralfate above 40% completely protected its activity.As the concentration of pepsin was 0.02mg/mL,the residuary antibody activity of IgY containing 10%,30%,50% sucralfate were 62.8%,71.6%,82.5% under Ph 2.0 and 75.6%,87.4%,94.5% under pH 3.0.After enforced with freeze thawing 10%,30%,50% sucralfate may make the residuary antibody activity of IgY increase to 70.6%,82.7% and 89.3%.Conclusion HpaA IgY containing 30% or above sucralfate can improve the tolerance of IgY against low pH,pepsin and freeze thawing intervention.It shows that sucralfate is a satisfactory protectant for HpaA IgY.
出处
《中国医药指南》
2011年第6期5-7,共3页
Guide of China Medicine
基金
重庆市科委(CSTC
2005EA5020)
