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Studies on Expression of Human-lactoferrin cDNA under the Multiple Promoter/enhancer of Bovine αs1 Casein/CMV

Author: HuangYuZheng
Tutor: ChengYong
School: Yangzhou University
Course: Clinical Veterinary Medicine
Keywords: CMV Mammary gland bioreactor Bovine αs1- casein Human lactoferrin Transgenic mice
CLC: Q78
Type: Master's thesis
Year: 2007
Downloads: 71
Quote: 3
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Abstract


Transgenic animal technology, preparation of high expression of human lactoferrin mammary gland bioreactor pursued the goal of the scientific community. Transgenic animal mammary gland bioreactor to obtain high-quality, low-cost medicinal recombinant human lactoferrin possible. However, human lactoferrin in the mammary gland of transgenic animals are still many at a low level of expression. To this end, we construct a pair of mammary specific expression vector, to prepare a transgenic mouse, by comparing the two different components (i.e., whether the sub-portions containing the CMV enhancer), and addition of CMV enhancer sub improving the human lactoferrin breast specific sexual expression levels; groping and screened at the individual level to the high expression of individual transgenic mammary gland bioreactor to produce transgenic sheep, and the high expression of the individual to lay the foundation. Of this study is to build a selection of bovine αs1-casein regulatory sequences, including long 2264bp5 'end regulatory region, the first part contains the second exon initiating ATG, intron 1, exon 1, 5' upstream the 818bp sequence; total length of 1440bp 3 'end of the regulatory region, including the final outside exons (18 exons) part of the last intron (intron 18), 3' untranslated region; another upstream splicing The ATG's rich of 4kb part, from -2264 bp to -6264 bp at. The specific sequence of GENBANK (accession number X59856). By Xhol digested the PG-LF plasmid (preserved) hLF cDNA fragment (approximately 2.3kb) work together to build a Bovine αs1-casein 5 'end of hLF was cDNA bovine αs1-casein 3' end to form the human lactoferrin cDNA mammary specific expression vector AF; in AF build based on the enhancement of CMV promoter portion-containing DNA fragment was cut from plasmid pCEP4 vector, enhanced promoter fragment hLF cDNA by bovine αS1-casein 5 'end of the bovine alpha S1-casein 3 'end of the build adult lactoferrin cDNA mammary specific expression vector AP. AP plasmid with Sal1 and Not1 restriction enzyme digestion, purified the approximately 11kb fragments of the eukaryotic expression vector. Optional C57 ♂ ICR ♀ 1491 mating, fertilized eggs, pronuclear microinjection method the expression vector fragment was introduced into mouse fertilized eggs male pronucleus and survival after injection of 630 of the number of eggs (graft number), the number of transplant recipients 37 only, the receptor pregnancy number 13, get born 51 pups, the fertilized egg survival rate was 42.3%; the AF components plasmid is treated the same way, to produce transgenic mice, 851 fertilized eggs by pronuclear The injection of the expression vector fragment into mouse zygotes male pronuclei survival after injection, the number of eggs (transplant) 490, the number of transplant recipients 20 receptor pregnancy number 15 and 56, get born offspring, the fertilized egg survival rate of 57.6%. 2 AP mice by PCR and PCR products were sequenced integrated exogenous gene (♀ 46 #, ♀ 42 #), transgene integration was 3.9%. Transgenic primary mouse and normal mouse mating F1 generation integrated mouse 5 F2 generation integrated mouse 7 human lactoferrin transgenic mouse strains. By enzyme-linked immunosorbent assay (ELISA assay) and Western blot (Western Blot) in the milk of transgenic mice and their offspring AP primary expression levels were detected. ELISA test results show that the primary transgenic mice and F1 and F2 generation of the milk of human lactoferrin expression were positive, which represent up to about 3 6 g · L original: AP -1 . Western Blot analysis that is consistent with standard human lactoferrin leucorrhea bands: the expression of transgenic mouse milk. In addition, the transgenic mice blood, The ELISA showed tear, constructed expression vector with breast specificity. AF transgenic mice milk collection, sent to save testing, test results Laboratory of Jiangsu Province, Institute of Nuclear Medicine. Screening by inbreeding, get AP homozygous transgenic mice (♀ 38 #, and ♂ 36 #), prolactin for mouse milk ELISA detection of the milk of human lactoferrin higher expression levels. While taking advantage of the laboratory has obtained PCL25 transgenic mice (♀ #), screening for homozygous mice (♀ 18 #), befriending distant of the two strains, screened to obtain double heterozygous mice, to improve the preparation of transgenic animals efficiency provide a new idea. Comparing the AF of the two members with the AP, the latter on the basis of the former increases the CMV enhancer; individual expression levels in transgenic mice, which was significantly higher than the former. The test results confirmed our vision, that is, cattle αs1-casein regulatory sequences and the CMV enhancer recombination promoter to build mammary specific expression vector, can greatly enhance the expression of human lactoferrin cDNA. The experiments also show that, the human lactoferrin breast we build good breast tissue specific expression vector expressing specific. At the same time, turn the screening of the gene in mice homozygous produce transgenic animals for us to shorten the cycle can also provide a theoretical basis.

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