Jeong Kyoung Yong, Yi Myung-Hee, Jeong Kyoung-jin, Lee Haeseok, Hong Chein-Soo, Yong Tai-Soon
Department of Environmental Medical Biology and Institute of Tropical Medicine, Arthropods of Medical Importance Resource Bank, Seoul, Korea.
Int Arch Allergy Immunol. 2009;148(4):339-45. doi: 10.1159/000170388. Epub 2008 Nov 11.
The cockroach allergen Bla g 4, a putative lipocalin, is known to exhibit frequent sequence variations. However, the previously reported cDNA sequences are truncated at the N terminus. This study was undertaken to investigate the mechanisms by which these sequence variations are generated.
Rapid amplification of cDNA ends PCR and RT-PCR were performed to obtain the full sequence of the Bla g 4 cDNA, and PCR was also used to clone the Bla g 4 genomic DNA. In addition, Bla g 4 protein variants were analyzed by two-dimensional gel electrophoresis.
Nine additional amino acid residues at the N terminus of Bla g 4 were identified, and 2 genes encoding Bla g 4, both of which consisted of 5 exons, were cloned. Examination of 34 clones of Bla g 4 cDNA obtained by RT-PCR revealed 14 variants. In particular, Bla g 4 sequences showed frequent clusters of variations in residues 38-45, 61-82 and 144-163. Differences in cDNA sequences may imply that RNA sequences are edited after transcription. More than 10 spots were identified between pH 5 and 7 upon two-dimensional gel electrophoresis, indicating that multiple variants of Bla g 4 are produced at the protein level.
Genetic polymorphisms among individual cockroaches, the existence of multiple genes and sequence variations caused by RNA editing produce sequence diversity of Bla g 4, which may influence its allergenicity. The sequence information obtained in this study will be helpful for the standardization of the cockroach allergen and thereby aid in the development of diagnostics and immunotherapeutics.
蟑螂过敏原Bla g 4是一种假定的脂质运载蛋白，已知其序列存在频繁变异。然而，先前报道的cDNA序列在N端是截断的。本研究旨在探讨这些序列变异产生的机制。
采用cDNA末端快速扩增PCR和逆转录PCR获得Bla g 4 cDNA的全长序列，同时利用PCR克隆Bla g 4基因组DNA。此外，通过二维凝胶电泳分析Bla g 4蛋白变体。
在Bla g 4的N端鉴定出另外9个氨基酸残基，克隆了2个编码Bla g 4的基因，二者均由5个外显子组成。对通过逆转录PCR获得的34个Bla g 4 cDNA克隆进行检测，发现14种变体。特别是，Bla g 4序列在第38 - 45、61 - 82和144 - 163位残基处频繁出现变异簇。cDNA序列的差异可能意味着RNA序列在转录后被编辑。二维凝胶电泳在pH 5至7之间鉴定出10多个斑点，表明在蛋白质水平上产生了多种Bla g 4变体。
个体蟑螂之间的遗传多态性、多个基因的存在以及RNA编辑导致的序列变异产生了Bla g 4的序列多样性，这可能会影响其致敏性。本研究获得的序列信息将有助于蟑螂过敏原的标准化，从而有助于诊断和免疫治疗的发展。
