海南医学
海南醫學
해남의학
HAINAN MEDICAL JOURNAL
2014年
22期
3277-3279,3280
,共4页
李秀丽%谷芳%姚元庆%刘忠宇
李秀麗%穀芳%姚元慶%劉忠宇
리수려%곡방%요원경%류충우
VEGF111b%pcDNA3.1%真核表达%多克隆抗体
VEGF111b%pcDNA3.1%真覈錶達%多剋隆抗體
VEGF111b%pcDNA3.1%진핵표체%다극륭항체
VEGF111b%pcDNA3.1%Karyotic expression%Polyclonal antibody
目的：克隆VEGF111b基因，构建真核表达载体pcDNA3.1-VEGF111b，并进行测序，制备VEGF111b多克隆抗体。方法丝裂霉素C诱导处理卵巢癌SKOV3细胞24 h，设计VEGF111b酶切引物，以SKOV3细胞的cDNA为模板，通过PCR得到VEGF111b特异性基因产物，克隆入真核表达载体pcDNA3.1中，获得重组真核表达载体pcDNA3.1-VEGF111b，测序验证。用KLH耦联VEGF111b特异性短肽，制备多克隆抗体，Western blot检测其特异性。结果成功扩增了VEGF111b基因，双酶切、测序鉴定证实目的基因成功克隆到真核表达载体pcDNA3.1中，测序结果与预测完全一致，成功制备了VEGF111b多克隆抗体，使用其多克隆抗体，Western blot能够检测到VEGF111b对应分子量的蛋白条带。结论本研究鉴定了pcDNA3.1-VEGF111b真核表达载体，并验证了VEGF111b多克隆抗体的特异性，为进一步研究VEGF111b蛋白的功能奠定了基础。
目的：剋隆VEGF111b基因，構建真覈錶達載體pcDNA3.1-VEGF111b，併進行測序，製備VEGF111b多剋隆抗體。方法絲裂黴素C誘導處理卵巢癌SKOV3細胞24 h，設計VEGF111b酶切引物，以SKOV3細胞的cDNA為模闆，通過PCR得到VEGF111b特異性基因產物，剋隆入真覈錶達載體pcDNA3.1中，穫得重組真覈錶達載體pcDNA3.1-VEGF111b，測序驗證。用KLH耦聯VEGF111b特異性短肽，製備多剋隆抗體，Western blot檢測其特異性。結果成功擴增瞭VEGF111b基因，雙酶切、測序鑒定證實目的基因成功剋隆到真覈錶達載體pcDNA3.1中，測序結果與預測完全一緻，成功製備瞭VEGF111b多剋隆抗體，使用其多剋隆抗體，Western blot能夠檢測到VEGF111b對應分子量的蛋白條帶。結論本研究鑒定瞭pcDNA3.1-VEGF111b真覈錶達載體，併驗證瞭VEGF111b多剋隆抗體的特異性，為進一步研究VEGF111b蛋白的功能奠定瞭基礎。
목적：극륭VEGF111b기인，구건진핵표체재체pcDNA3.1-VEGF111b，병진행측서，제비VEGF111b다극륭항체。방법사렬매소C유도처리란소암SKOV3세포24 h，설계VEGF111b매절인물，이SKOV3세포적cDNA위모판，통과PCR득도VEGF111b특이성기인산물，극륭입진핵표체재체pcDNA3.1중，획득중조진핵표체재체pcDNA3.1-VEGF111b，측서험증。용KLH우련VEGF111b특이성단태，제비다극륭항체，Western blot검측기특이성。결과성공확증료VEGF111b기인，쌍매절、측서감정증실목적기인성공극륭도진핵표체재체pcDNA3.1중，측서결과여예측완전일치，성공제비료VEGF111b다극륭항체，사용기다극륭항체，Western blot능구검측도VEGF111b대응분자량적단백조대。결론본연구감정료pcDNA3.1-VEGF111b진핵표체재체，병험증료VEGF111b다극륭항체적특이성，위진일보연구VEGF111b단백적공능전정료기출。
Objective To clone the VEGF111b gene, construct eukaryotic expression system pcD-NA3.1-VEGF111b, sequence VEGF111b gene, and prepare VEGF111b polyclonal antibody. Methods With VEGF111b-specific primers and RT-PCR, VEGF111b gene was amplified from ovarian cancer SKOV3 cells treated with mitomycin C for 24 h. PCR product was cloned into eukaryotic expression vector pcDNA3.1 to construct pcD-NA3.1-VEGF111b, and was then sequenced. VEGF111b-specific peptide was connected KLH to prepare the polyclonal antibody. Western blot was used to detect its specificity. Results VEGF111b gene was successfully amplified by RT-PCR and it was proved to be cloned into pcDNA3.1 vector by double digestion with restriction endonuclease and se-quencing analysis. We have prepared VEGF111b polyclonal antibody successfully, and detected protein band of the corresponding molecular weight using the polyclonal antibody by Western blotting. Conclusion This study not only identified eukaryotic expression vector pcDNA3.1-VEGF111b, but also verified the specificity of VEGF111b poly-clonal antibody, which provides a basis for further research on VEGF111b function.
