Cloning of sheep FGF5 gene and construction of lentiviral expression vector
A technology of lentiviral vector and expression vector, which is applied in the field of full-length cDNA sequence cloning, and can solve the problems of no variant splicing body of sheep FGF5 and lack of sheep, etc.
Active Publication Date: 2013-02-27
新疆维吾尔自治区畜牧科学院中国-澳大利亚绵羊育种研究中心
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Problems solved by technology
[0005] Due to the lack of the sequence of sheep FGF5 at home and abroad, the variant splice form of sheep FGF5 has not been found
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[0030] The present invention is further described with reference to examples.
[0031] (1) Experimental sample collection and total RNA extraction
[0032] The fine-wool sheep skin tissue was collected, put into cryopreservation tubes and quickly stored in liquid nitrogen for future use. Take 100 mg tissue sample and grind it in liquid nitrogen, then extract according to the instruction of Hangzhou Bioer RNA extraction kit.
[0033] (2) RT-PCR
[0034] According to the instruction manual of reverse transcriptase M-MLV (Bao Biology), the RNA of sheep skin tissue was reverse-transcribed, and the reverse transcription system was referred to the system in the instruction manual.
[0035] (3) Primer design
[0036] Use Oligo6.0 to design and amplify exons 1 and 3, clone them into T vectors and send them to Shanghai Bioengineering Company for sequencing. The sequencing results are shown in Sequence Tables 1 and 2; design amplification based on the sequencing results of exons 1 an...
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The invention discloses the cloning of a sheep FGF5 gene and the construction of a lentiviral expression vector. A method comprises the following steps of: determining a complete sequence of a coding region of a sheep Noggin gene; directionally cloning target genes to a pLEX lentiviral expression vector to obtain lentiviral expression vectors of FGF5 and FGF5S genes, producing recombinant viruses, infecting 293T cells, and verifying the expression of a recombinant protein, wherein a primer for amplifying an exon 1 is provided, a primer for amplifying 5-RACE is provided, a primer for amplifying an exon 3 is provided, a primer for amplifying 3-RACE is provided and is designed according to a sequencing result of the exon 3, and a primer for amplifying the full length of FGF5 is designed according to sequencing results of the 5-RACE and the 3-RACE, namely a full-length complementary deoxyribonucleic acid (cDNA) sequence of the FGF5 is amplified and full-length sequences of the FGFT and FGF5S are determined through sequencing; and cloning and sequencing, namely recovering and purifying an amplification product of the sheep FGF5 gene, connecting the amplification product with a PMD-18T vector by utilizing T4DNA ligase, transforming E. coli DH5alpha, screening by an ampicillin plate, identifying by a polymerase chain reaction (PCR) method and a restriction enzyme digestion method, and finishing the sequencing of positive clones by Shanghai bioengineering Co., Ltd..
Description
technical field [0001] The present invention relates to the full-length cDNA sequence cloning of sheep FGF5 (fibroblast growth factor 5) gene coding region (CDS). Background technique [0002] FGF5 is a regulatory factor that plays an important role in regulating the growth cycle of hair follicles. In 1994, Hebert 【1】 Using embryonic stem cell gene knockout technology, it was reported for the first time in the internationally renowned Cell magazine that the coat length of homozygous mice with FGF5 gene knockout is 50% longer than that of heterozygotes. The gene is the allele of the FGF5 mutation. FGF5 is mainly expressed in the outer root sheath of hair follicles. Early studies have shown that the reason for the long coat of Angora rats is due to the prolonged anagen phase of the hair follicles of the mutant mice. Zhan (1988) 【2】 The fifth fibroblast growth factor was found in human tumor cells, and it was named FGF5. There are two variant splicing forms, encoding 122 an...
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IPC IPC(8): C12N15/12
Inventor 刘明军贺三刚李文蓉张雪梅张宁
Owner 新疆维吾尔自治区畜牧科学院中国-澳大利亚绵羊育种研究中心