rhPA/GH双转基因兔的制备及rhPA表达检测  

Preparation and Expression of rhPA/GH Double Transgenic Rabbits

作　　者：宋绍征 于康英 张婷[2] 陆睿 潘生强 成勇[2] 周鸣鸣 SONG ShaoZheng;YU KangYing;ZHANG Ting;LU Rui;PAN ShengQiang;CHENG Yong;ZHOU MingMing(School of Nursing,Wuxi Taihu University,Wuxi 214000,Jiangsu;Jiangsu Provincial Research Center for Animal Transgenesis and Biopharming/College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,Jiangsu)

机构地区：[1]无锡太湖学院护理学院基础医学系,江苏无锡214064 [2]扬州大学兽医学院/江苏省转基因动物制药工程研究中心,江苏扬州225009

出　　处：《中国农业科学》2021年第2期412-421,共10页Scientia Agricultura Sinica

基　　金：江苏省高校自然科学基金面上项目(19KJB180030);国家转基因生物新品种培育重大专项(2014ZX08008-004)。

摘　　要：【目的】双基因共整合可协同促进转基因生物的目的基因表达水平提高,研究获得rhPA/GH双转基因兔,并比较分析目的基因rhPA的表达水平及兔个体生长发育情况,为制备高表达rhPA转基因动物和遗传育种提供新思路。【方法】利用NotⅠ/SalⅠ双酶切PCL25/GH质粒和QIAGEN DNA胶纯化试剂盒回收显微注射用基因片段。以3只rhPA单转基因兔(标号K06、K10、K17)作为供体兔,通过FSH/hCG超数排卵、受精卵原核显微注射、胚胎移植、苯酚/氯仿抽提新生仔兔耳尖组织基因组、PCR整合检测等方法获得rhPA/GH双转基因兔。经ELISA和Western blotting对转基因兔乳清进行表达检测,比较分析单、双基因表达rhPA水平。测量不同月龄的转基因兔体重,通过监测双转基因兔不同生长阶段的体重来分析GH对兔生长发育的影响。【结果】成功获得了约16700 bp大小的显微注射用基因片段,共超排3只供体兔获得122枚卵,其中103枚受精卵,受精率为84.4%(103/122),显微注射后挑取形态较好的81枚移植6只同步发情受体兔,5只兔怀孕,妊娠率为83.3%(5/6),妊娠到期共出生32只仔兔。通过PCR检测鉴定有19只携带rhPA基因的转基因兔,其中有11只rhPA/GH双转基因兔(7♂,4♀),双基因整合率为34.4%(11/32),且4只rhPA/GH双转基因母兔来源亲代分别为K06供体兔2只(标号K06-1、K06-2)、K10供体兔1只(标号K10-1)、K17供体兔1只(标号K17-1)。K06号rhPA单转基因兔乳清中rhPA表达量为42.2μg·mL^-1,K06-1和K06-2号rhPA/GH双转基因兔乳清中rhPA表达量分别为432、444μg·mL^-1;K10号rhPA单转基因兔乳清中rhPA表达量为42.8μg·mL^-1,K10-1号rhPA/GH双转基因兔乳清中rhPA表达量为636μg·mL^-1;K17号rhPA单转基因兔乳清中rhPA表达量为15.2μg·mL^-1,K17-1号rhPA/GH双转基因兔乳清中rhPA表达量为248μg·mL^-1。4只rhPA/GH双转基因母兔(K06-1、K06-2、K10-1、K17-1)乳腺表达rhPA含量在248-636μg·mL^-1之间,远远高于rhP【Objective】The integration of the double genes is able to promote the expression level of the target genes in the transgenic organisms.The aim of this study was to obtain the rh PA/GH double transgenic rabbits,and then the expression level of the target gene rhPA and the individual growth and development of these rabbits were compared and analyzed,so as to provide a new new approach for preparation of high expression rhPA transgenic animals and genetic breeding.【Method】NotⅠ/SalⅠdouble enzyme digestion PCL25/GH plasmid and QIAGEN DNA gel purification kit were used to recover gene fragments for microinjection.The three rhPA single-transgenic rabbits(K06,K10 and K17)were used as donors.The rhPA/GH double-transgenic rabbits were obtained by FSH/hCG superovulation,pronuclear microinjection of fertilized eggs,embryo transfer,phenol/chloroform extraction of newborn rabbit’ear tip tissue genome and PCR integrated detection.In addition,the expression levels of rhPA in single-transgenic rabbit and double-transgenic rabbit whey were compared by ELISA and Western blotting.The body weight of transgenic rabbits at different months was measured,and the effects of GH on growth and development of rabbits were analyzed by body weight at growth stage.【Result】The about 16700 bp of microinjection gene fragments were successfully obtained.A total of 122 eggs were obtained from 3 donor rabbits,103 of which were fertilized,and the fertilization rate was 84.4%(103/122).After microinjection,81 fertilized eggs with good morphology were selected and transplanted into 6 recipient rabbits by synchronous estrus.Five rabbits were pregnant and the pregnancy rate was 83.3%(5/6).A total of 32 offspring were born at the end of pregnancy.There were 19 rhPA transgenic rabbits identified by PCR,and 11 of them were the rhPA/GH double transgenic rabbits(7♂,4♀),so the double-gene integration rate was 34.4%(11/32).The four rhPA/GH double-transgenic female rabbits were derived from K06 donor rabbits(labelled K06-1 and K06-2),K10 donor

关 键 词：双转基因兔 表达 超数排卵 显微注射 生长曲线 

分 类 号：S829.1[农业科学—畜牧学]