植物抗体基因工程：II．马铃薯Y病毒中和抗体cDNA基因文库的构建及含抗体轻链和重链基因阳性克隆的筛选

Antibody Genetic Engineering in Plant:II .Construcring and Screening of cDNA Gene Library of Mouse Monoclonal Antibody against Potato Virus Y

从大量繁殖的Ｄ７３杂交瘤细胞中提取制备其基因组ｍＲＮＡ，并以此为模板，经反转录途径获得基因组ｃＤＮＡ，随后将其插入到λｇｔ１１中，构建了马铃薯Ｙ病毒小鼠单克隆抗体ｃＤＮＡ基因文库。经免疫原位杂交，从该基因文库中筛选出３０个含抗体轻链基因和１０个含抗体重链基因的阳性克隆，进一步将其插入到ｎＧＥＭ－７Ｚ（＋）质粒中，经酶切分析和电泳检测，证实在获得的轻链和重链阳性克隆中各有一个质粒（Ｋ６和Ｇ９）具有较长的外源ＤＮＡ插入（分别为１Ｋｂ和１．６Ｋｂ），它们可能编码完整的抗体轻链和重链蛋白。

A cDNA library was constructed in λgt11 vectors,complementary to the mRNA isolated from a mousehybridoma(D73)raised against potato virus Y(PVY).Thirty cDNA clones containing the light chain genesequence and ten cDNA clones containing heavy chain gene sequence were obtained from the cDNA Iibraryby in situ immunohyubidization with goat anti-mouse kappa and Y1-chain specific antibody conjugated toaikaline phosphatase,among which the clone K6(light chain)and G9(hveay chain)were likely torepresent full transcripts of light and heavy chain genes,respectively,on the grounds of their sizes andrestriction maps.