Preparation method of veterinary follicle-stimulating hormone analogue
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A technology of follicle stimulating hormone and analogues, applied in the field of genetic engineering, can solve problems such as not fully considering the influence of connection sequences, and achieve significant commercial value, good substitution effect, and increased expression
Inactive Publication Date: 2014-11-19
林静静 +1
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This invention emphasizes the effect of glycosylation on long-acting effects, but does not fully consider the influence of the connecting sequence itself on the expression of the fusion protein
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Embodiment 1
[0024] Example 1: Construction of expression plasmids and establishment of engineered cells
[0025] 1. According to the amino acid sequence and nucleotide sequence of natural sheep FSH recorded on Genbank ( http: / / www.ncbi.nlm.nih.gov / sites / entrez? cmd=Retrieve&db=nucleotide &dopt=GenBank&list_uids=57619323 ,and http: / / www.ncbi.nlm.nih.gov / sites / entrez? cmd=Retrieve&db=nucleotide &dopt=GenBank&list_uids=1365 ), design and synthesize the whole gene SEQ ID NO: 1, containing the known linker sequence (Linker1): ggatccggct ccaacgccac cggctccggc tccaacgcca cctccggctc cactag sequence (J Clin Endocrinol Metab, 2004, 89: 5204-5212).
[0026] 2. Construction of expression plasmids: the double-stranded SEQ ID NO 1 and pcDNA3.1 (+) plasmids were respectively subjected to double enzyme digestion (NheI and EcoRI), separated by 1% agar electrophoresis, and extracted. The SEQ ID NO 1 and pcDNA3.1(+) plasmids treated with double digestion were ligated by ligase. By standard CaCl 2...
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Abstract
The invention provides a veterinary follicle-stimulating hormone analogue and a preparation method thereof. A connecting sequence is designed according to length, foldability, hydrophobicity and charge effect of a connecting sequence between an alpha peptide chain and a beta peptide chain, the corresponding nucleotide sequence of the connecting sequence is any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20, and respective expression quantity and biological activity are compared by virtue of the study. A fused protein of the prepared veterinary follicle-stimulating hormone analogue is similar to the known fused protein in term of biological functions, the expression quantity of multiple fused proteins is obviously increased, the veterinary follicle-stimulating hormone analogue can be used for replacing natural ovarian-stimulating hormone, has great commercial value and is worthy of wide-range popularization.
Description
technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for preparing a veterinary follicle stimulating hormone analogue. Background technique [0002] Follicle-stimulating hormone (FSH) is a glycoprotein hormone synthesized and secreted by basophilic cells of the anterior pituitary gland of animals. keys are connected. It can promote the secretion of estrogen, induce estrus in animals, promote the maturation of follicle eggs, superovulation and stimulate sperm production. [0003] At present, FSH for veterinary use is all animal brain tissue extracts, not only doped with different levels of luteinizing hormone, resulting in unstable super-expelling effect, but also has the potential risk of carrying biologically derived infectious agents. The development of veterinary FSH through genetic engineering is a current research hotspot. There are two main genetic engineering strategies for gene recombination of FSH that have b...
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