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Recombined human growth hormone gene bacilliform virus, preparation and application thereof

A technology of human growth hormone and silkworm baculovirus, applied in biochemical equipment and methods, botany equipment and methods, applications, etc., can solve the problem of loss of activity, achieve high expression efficiency and reduce production costs

Inactive Publication Date: 2014-05-21
浙江中奇生物药业股份有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, HGH is widely used in hormone replacement therapy, but because it is easily metabolized and inactivated in the intestinal tract, the HGH preparations on the market are mainly injections

Method used

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  • Recombined human growth hormone gene bacilliform virus, preparation and application thereof
  • Recombined human growth hormone gene bacilliform virus, preparation and application thereof
  • Recombined human growth hormone gene bacilliform virus, preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1. Amplification of HGH gene

[0036] A pair of primers were designed using the HGH gene as a template. The upstream primer contained an EcoR I restriction site, and the downstream primer contained a Sac I restriction site. The primers were designed as follows:

[0037] Upstream primer P1: 5'GGGAATTCAGTTTCCTAC-TATACCAC 3' (SEQ NO.2);

[0038] Downstream primer P2: 5'TTGAGCTCCTAGAAGCC-ACAGCTGCC 3' (SEQ NO.3).

[0039] Human growth hormone HGH gene was amplified by PCR from the recombinant plasmid PWR-HGH (gifted by Mr. Wu Xiangfu, Shanghai Institute of Biochemistry). Using the recombinant plasmid PWR-HGH as a template, pre-denaturation at 94°C for 5 minutes, followed by denaturation at 94°C for 1 minute; annealing at 57°C for 1 minute; extension at 72°C for 1 minute, 30 cycles; Amplify the target HGH gene fragment.

Embodiment 2

[0040] Example 2. Construction of pAcHLT-HGH vector containing HGH gene

[0041] The HGH gene fragment cut by EcoR I and Sac I was ligated by T4 DNA enzyme (MBI) and cloned into the pAcHLT-B baculovirus transfer vector (purchased from BD Biosciences Pharmingen) by EcoR I and Sac I, so that HGH The gene is placed under the control of the polyhedrin (polyhedrin, ph) gene promoter, and the recombinant transfer plasmid pAcHLT-HGH (such as figure 1 ), the gene sequence was confirmed to be correct by restriction analysis and sequencing. In the silkworm expression system, the recombinant protein HGH was expressed in the form of fusion protein.

Embodiment 3

[0042] Embodiment 3. Preparation of Bombyx mori recombinant baculovirus BmBacHGH

[0043] Take 5 μl of recombinant transfer plasmid pAcHLT-HGH DNA and 6 μl of linearized virus BmBacPAK6 DNA (purchased from Shanghai Biochemical Cell Institute), add 100 μl of serum-free TC-100 medium (purchased from GIBCOBRL Company) and mix well. Take 6 μl of Dosper (Bowringman Company) and add 100 μl of serum-free TC-100 medium and mix well. BmN cells (purchased from Shanghai Biochemical Cell Institute) previously cultured in a 35mm plate were washed twice with serum-free TC-100 medium, and the pAcHLT-HGH transfer plasmid and Dosper mixture were added dropwise, and incubated at 27°C for 4-5 On the next day, the supernatant was collected for the first round of plaque screening. Take 5 μl of the supernatant to infect the Bm N cells in a 35mm plate, discard the supernatant after 1 hour and add an equal amount of mixed TC-100 medium and low melting point agarose. After 4-5 days, plaques were pic...

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Abstract

The invention relates to a recombinant baculovirus and a preparation method as well as applications thereof. The invention constructs the recombinant baculovirus with a human growth hormone gene HGH, recombinant HGB protein can be effectively expressed in a domestic silkworm, and the drug experiment proves that recombinant HGB protein products have no toxic side effect in animal bodies but have the advantages of safety and reliability, good effect of oral application and absorption as well as anti-aging performance. The manufacturing material of the recombinant baculovirus is easy to be obtained, and the manufacturing cost is low, so the recombinant baculovirus has important industrial value and broad prospect of being taken as oral drug and food.

Description

technical field [0001] The invention relates to the technical field of polypeptide medicine produced by genetic engineering in biotechnology pharmaceutical engineering. Background technique [0002] Human Growth Hormone (HGH, Human Growth Hormone) is a polypeptide secreted by the human body itself. It is a single-chain globular protein composed of 191 amino acid residues, with a molecular weight of 22,000 Daltons. It is secreted by the pituitary gland and passed through the Released by the thalamus, it controls growth and development, immunity and human metabolism. HGH stimulates the differentiation and proliferation of epiphyseal chondrocytes, stimulates the growth of cartilage stromal cells, causes linear growth acceleration and bone widening; regulates growth hormone deficiency or adult fat, bone metabolism, heart and kidney function, and improves exercise capacity and quality of life. [0003] HGH can promote the decomposition of fat in adipose tissue and increase plasm...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/01C12N15/18C12N15/63A01K67/04A61K38/27A61K9/19A61K47/46A61P39/06
Inventor 任飞杨昆陈国刚何春波
Owner 浙江中奇生物药业股份有限公司
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