【摘要】 以猪垂体ｍＲＮＡ为模板，Ｏｌｉｇｏ（ｄＴ）（１２～１８）为反转录引物，在ＡＭＶ－ＲＴ的催化下，先合成ｃＤＮＡ第一链，再以ＲＮａｓｅＨ降解的ｍＲＮＡ小分子为引物、ｓｓ－ｃＤＮＡ为模板，在Ｅ．ｃｏｌｔＤＮＡＰｏｌ．Ｉ的催化下合成ｃＤＮＡ第二链，并用Ｅ．ｃｏｌｉＤＮＡｌｉｇａｓｅ连接缺口和Ｔ４ＤＮＡＰｏｌ补齐末端，以获得长度完整的ｄｓ－ＣＤＮＡ分子。根据已知的ｐＧＨ基因序列，设计并合成了一对特异引物分子，以总ｃＤＮＡ为模板，进行ＰＣＲ扩增，分子杂交证明，获得的扩增片段即为侧翼带有重组位点和附加密码的编码ｐＧＨ成熟肽的ＤＮＡ序列。更多还原

【Abstract】 Using oligo (dT)12～18as primer and mRNA from porcine pituitary gland as template, the first strand of cDNA was synthesized by AMV reverse transcriptase; and then the second strand of cDNA was replicated by E. colt DNA polymerase I after cutting mRNA into oligonucleotides with RNaseH. The full-length of double strand cDNA was thus obtained with further treatment by E. colt DNA ligase and T. DNA polymerase. On the basis of the known DNA sequence for porcine growth hormone, a pair of specific primers were designed and synthesized. Subsequently, polymerase chain reaction for amplifying the sequence encoding mature porcine growth hormone was conducted using these primers and synthesized template. Electrophoresis and southern blotting showed that the amplified fragment by PCR was the unique DNA sequence of mature porcine growth hormone,flanked by designed restriction site and codon sequences.更多还原