从人活化B细胞株3D5细胞λgtll cDNA文库筛选到的1529bp cDNA的核苷酸序列分析

Sequencing of a l529bp cDNA screened from theλgtll cDNA library constructed from a human activated B cell 3D5

对用单抗５Ｃ５和单抗５Ｃ５－Ｇ１的混合物从３Ｄ５细胞λｇｔｌｌｃＤＮＡ文库筛选到的７个阳性ｃＤＮＡ克隆中的１个（３Ｄ５－５）进行了核苷酸序列分析。由于３Ｄ５－５ｃＤＮＡ长１．６ｋｂ左右，不能直接测序，故先依测序用质粒ｐＢＳｃｒｉｐｔ－ＫＳ多克隆位点中的内切酶点行单酶切，经电泳分析画出测序用的物理图谱，再用相应内切酶行单酶酶切。藉低熔点琼脂糖回收各个片段。带质粒ｐＢＳｃｒｉｐｔ－Ｋ５的大片段经直接自身环化，小片段与质粒载体ｐＢＳｃｒｉｐｔ－ＫＳ重组，构建测序用的亚克隆。用双链双脱氧末端终止法测各亚克隆的核苷酸序列，再拚接出全长为１５２９ｂｐ的３Ｄ５－５ｃＤＮＡ全长序列。与国际基因库资料比较，未见有相同的序列。此ｃＤＮＡ中有一个长４６５ｂｐ（从５４０ｂｐ－１００４ｂｐ）的ＯＲＦ，编码１５４个氨基酸的蛋白质。此蛋白质中有二个疏水区（第１～３４位氨基酸及第７９～１０９位氨基酸），提示它可能属Ⅰ类穿膜蛋白。

One (3D5-5) of the seven cDNA clones screened from a 3DS cell λgtll cDNA library with a mixture of McAb SC5 and SC5-Gl has been sequenced Since the 1.6bp cDNA can not be sequenced directly , an endonuclease map of the cDNA was detected first. The cDNA was inserted into the plasmid pBScript- KS after being cut with EcoR I and Not I . The recombinants were cut with each endonuclease which occupies one point in the multiple cloning site in the plasmid. On the electrophoresis the length of each fragment can be measured, and then a physical map of the 3D5-5 cDNA for sequencing can be drawn. 4 relevant subclones were developed by means of recirculation after being cut with Cla I , HincⅡ , Pst I and Sal I , and 5 other subclones were constructed through recombination of the small fragments produced by Cla I , HincⅡ and Pst I with the plasmid. The nucleotide sequences of them were detected by dideoxynucleotide chain termination method. The length of the 3D5-5 cDNA is 1529bp , which is not homologous with any cDNA in the Genbank in the world. There exists an 4 65bp long open reading frame (ORF) in the cDNA from 540bp to 1004bp which codes a 154 aa protein. Two hydrophobic transmembrane regions from 1 tb 34 aa and from 79～109 aa are found in the protein ,suggesting a class I integral membrane protein. The 1529 cDNA has been accepted by GenBank with an accession number of U40710. .