Prevention of mite allergy by oral administration of Lactobacillus transformed with expression vector carrying Derf2-cDNA.

为了预防尘螨过敏的发生，我们试图通过口服经热处理的大肠杆菌或转化表达Derf2蛋白的乳酸菌活菌来诱导小鼠对尘螨过敏的免疫耐受。利用表达载体pUEX1在E.Coli或LL中表达DERF2-cDNA文库。用兔抗Derf2抗体对克隆进行免疫筛选。给C57BL或A/J小鼠口服表达或不表达DERF2的微生物(109次/天)或PBS，共10天。然后对小鼠进行免疫接种。重组DERF2免疫后第28天加强免疫。在加强免疫当天和第一次免疫后56天采集血样。用双抗体夹心法测定免疫球蛋白、免疫球蛋白、免疫球蛋白1抗体效价。出乎意料的是，口服表达大肠杆菌的Derf2蛋白可以促进小鼠的抗体形成，特别是IgE。大肠杆菌的佐剂活性可能是由于其内毒素或其他细胞壁物质所致。大肠杆菌不宜口服，而表达L1的Derf2对IgE、Ig G和Ig G1之间的抗体形成没有任何影响。在本研究中，我们未能成功地诱导小鼠口服耐受。其中一个问题是在乳酸菌中表达了少量的Derf2蛋白。目前，我们正在研究合适的乳酸菌种类、表达载体和DERF2-cDNA的插入条件。

To prevent the development of mite allergy we tried to induce immune tolerance to mite allergy in mice by oral administration of heat-treated E.coli or alive lactobacillus lactis(LL) which were transformed to express Derf2 protein. Derf2-cDNA library was expressed in E.coli or LL employing an expression vector pUEX1. The clones were screened colony immunoassay employing rabbit anti-Derf2 antibody . C57BL or A/J mice were given orally Derf2- expressing or non-expressing microorganisms (10^9/day) or PBS only for 10 days. Then mice were immunized i.p. with recombinant Derf2 and boosted at 28th day. Bloods were collected at the booster day and 56days after the first immunization. IgE, IgG and IgG1 antibody titer were assayed by ELISA method. Unexpectedly, an oral administration of Derf2 protein expressing E.coli enhanced antibody formation, especially IgE in mice. Adjuvant activity of E.coli was likely due to its LPS or other cell- wall substances. E.coli was suggested to be inappropriate for oral administration, On the other hand, Derf2 expressing LL did not show any change in the antibody formation among IgE, IgG and IgG1. In the present study we could not succeed in inducing the oral tolerance in mice. One of problems was a small amount of the expression of Derf2 protein in lactobacillus. At present we investigate the proper kinds of lactobacillus, expression vectors and the insertion conditions of Derf2-cDNA.