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

为了防止螨虫过敏的发生，我们尝试通过口服经热处理的大肠杆菌或转化为表达Derf2蛋白的活乳酸杆菌（LL）诱导小鼠对螨虫过敏的免疫耐受。利用表达载体pUEX1在大肠杆菌或LL中表达Derf2-cDNA文库。采用兔抗derf2抗体集落免疫法筛选克隆。C57BL或A/J小鼠只口服表达或不表达Derf2的微生物（10^9/天）或PBS，持续10天。然后用重组Derf2免疫小鼠，并在第28天进行强化免疫。分别于第1次免疫后第56天和第1次免疫后第56天采血。ELISA法检测血清IgE、IgG、IgG1抗体滴度。出乎意料的是，口服表达大肠杆菌的Derf2蛋白可增强小鼠体内的抗体形成，尤其是IgE。大肠杆菌的佐剂活性可能与它的LPS或其他细胞壁物质有关。另一方面，表达Derf2的LL在IgE、IgG和IgG1之间的抗体形成没有变化。在目前的研究中，我们无法成功地诱导小鼠口服耐受。其中一个问题是Derf2蛋白在乳酸菌中表达量少。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.