rhPA/GH双转基因兔的制备及rhPA表达检测

doi:10.3864/j.issn.0578-1752.2021.02.016

摘要/Abstract

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16 700 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15.2 μg·mL ^-1,K17-1号rhPA/GH双转基因兔乳清中rhPA表达量为248 μg·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rhPA单转基因母兔（K06、K10、K17）乳腺的表达含量（15.2-42.8μg·mL^-1）,表达水平显著提高了约10.2-16.3倍,说明GH能够协同促进目的基因rhPA在转基因兔乳腺中的表达水平。Western blotting结果显示出现一约39.0 kD大小的条带,与目的蛋白rhPA大小相同,进一步证明转基因兔乳腺中表达的这种蛋白为目标产物rhPA。对rhPA/GH双转基因兔从出生到6个月的体重进行连续测量,发现与正常非转基因兔的体重没有明显的差异,绘制生长曲线进一步表明4只整合GH的转基因兔与2只未整合GH的正常兔在生长发育的不同阶段体重上没有显著性差异,成长至6个月的体重均在4.0-5.0 kg之间,这证明GH的导入并不影响转基因兔的存活和正常生长发育至成年。【结论】成功地制备了rhPA/GH双转基因兔,并证明了GH基因的导入能够显著地提高目的基因rhPA的表达量,且不会对转基因兔的生长发育产生影响,这为将来制备高表达转基因兔及其它动物奠定了基础,也为转基因动物乳腺生物反应器和转基因育种建立提供了新思路、新方法。

关键词: 双转基因兔, 表达, 超数排卵, 显微注射, 生长曲线

Abstract:

【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 rhPA/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】 Not Ⅰ/ Sal Ⅰ double 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 16 700 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 rabbit (labeled K10-1) and K17 donor rabbit (labeled K17-1), respectively. The rhPA expression in single transgenic rabbit whey of No. K06 was 42.2 μg·mL ^-1, and the rhPA expression in the double transgenic rabbit whey of No. K06-1 and K06-2 was 432 and 444 μg·mL ^-1, respectively. The rhPA expression in the single transgenic rabbit whey of No. K10 was 42.8μg·mL^-1, and the rhPA expression in the double transgenic rabbit whey of No. K10-1 was 636 μg·mL ^-1. The rhPA expression in single transgenic rabbit whey of No. K17 was 15.2μg·mL^-1, and which in double transgenic rabbit whey of No. K17-1 was 248 μg·mL ^-1. The expression of rhPA in mammary glands of four female rhPA/GH double-transgenic rabbits (K06-1, K06-2, K10-1, and K17-1) were 248-636 μg·mL^-1, which was much higher than that of rhPA single-transgenic rabbits (K06, K10, K17, expression levels: 15.2-42.8μg·mL ^-1). That is to say, the expression level significantly increased about 10.2 to 16.3 times, and it showed that GH could synergistically promote the expression level of target gene rhPA in the mammary glands of transgenic rabbits. Besides, the western blotting results showed a band of about 39.0 kDa, which was the same size as the target protein rhPA, further proving that this protein expressed in the breast of transgenic rabbits was the target product rhPA. By measuring the weight of rhPA/GH double-transgenic rabbits from their birth to 6 months continuously, it was found that the weight was no significant difference between the rhPA/GH transgenic rabbits and the normal non-transgenic rabbits. The growth curve was drawn to further indicate that 4 transgenic rabbits with integrated GH and 2 normal rabbits without integrated GH had no significant difference in body weight at different stages of growth and development. The body weight for 6 months was about 4.0-5.0 kg. This proved that the introduction of GH did not have harmful influence on their survival and normal growth and development to adulthood. 【Conclusion】In this experiment, rhPA/GH double-transgenic rabbits were successfully prepared, and it has been proved that the introduction of GH could significantly increase the expression level of rhPA. Moreover, it could not affect the growth and development of transgenic rabbits. This laid a foundation for the preparation of high expression transgenic rabbits and other animals in the future, and also provided a new idea and method for the establishment of transgenic animal mammary gland bioreactors and transgenic breeding..

Key words: double transgenic rabbits, expression, superovulation, microinjection, growth curve

宋绍征,于康英,张婷,陆睿,潘生强,成勇,周鸣鸣. rhPA/GH双转基因兔的制备及rhPA表达检测[J]. 中国农业科学, 2021, 54(2): 412-421.

SONG ShaoZheng,YU KangYing,ZHANG Ting,LU Rui,PAN ShengQiang,CHENG Yong,ZHOU MingMing. Preparation and Expression of rhPA/GH Double Transgenic Rabbits[J]. Scientia Agricultura Sinica, 2021, 54(2): 412-421.

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引物名称 Primer name	引物序列 Primer sequence (5°-3°)
gGH-F	CCGCTCGAGCGGATGATGGCTGCAGGCCCCCGGA
gGH-R	CCGCTCGAGCGGCTAGAAGGCACAGCTGGCCTCCCCG
CMV/tPA-F	CGTGGATAGCGGTTTGA
CMV/tPA-R	GAGCCCTCCTTTGATGC

月龄Month	N1	N2	K06-1	K06-2	K10-1	K17-1
新出生Newborn	53	66	61	58	63	49
1	582	715	684	670	492	567
2	1416	1528	1432	1587	921	1384
3	2587	3199	2876	3022	2165	2455
4	3621	4342	4042	4111	3758	3469
5	4125	4752	4635	4425	4225	4021
6	4367	4967	4711	4698	4412	4232