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Construction method of transgenic chlamydomonas reinhardtii for expressing human tissue kallikrein

A technology of kallikrein and construction method, which is applied in the field of construction of transgenic Chlamydomonas reinhardtii, can solve the problems of lack of glycosylation modification, high culture cost, and inability to carry out stable genetics, etc. Rich and broad application prospects

Active Publication Date: 2012-01-25
深圳市微宇生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Escherichia coli belongs to prokaryotes, lacks the glycosylation modification of eukaryotes, and is prone to produce endotoxins, etc.; CHO cells are difficult to cultivate, the conditions are difficult to master, and the cultivation costs are high, making it difficult to apply industrially; insect cells have low sugar content. Kylation modification ability; in egg embryos, hK1 is only transiently expressed and cannot be stably inherited
The above deficiencies have greatly hindered the transformation of this technology into actual production.

Method used

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  • Construction method of transgenic chlamydomonas reinhardtii for expressing human tissue kallikrein
  • Construction method of transgenic chlamydomonas reinhardtii for expressing human tissue kallikrein
  • Construction method of transgenic chlamydomonas reinhardtii for expressing human tissue kallikrein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Selection and cultivation of transgenic recipient algal strains

[0047] Cell wall-defective Chlamydomonas reinhardtii (Chlamydomonas reinhardtii, cc-849, purchased from the Chlamydomonas Genetic Center of Duck University, USA) and cytochrome b-deficient Chlamydomonas reinhardtii (cc-2654, purchased from the Chlamydomonas Genetic Center of Duck University, USA) were selected as Transgenic recipient strains. Use TAP medium as the medium for Chlamydomonas reinhardtii, its formula and composition are as follows: 2.42g Tris, 25mL 4 × Beijerinck salts (16g NH 4 Cl, 2gCaCl 2 .2H 2 O, 4g MgSO 4 .7H 2 Dissolve O in water, dilute to 1L), 1mL 1M(K)PO 4 , 1mL Trace trace element mixture (11.4g H 3 BO 3 , 5.6g MnCl 2 .4H 2 O,22gZnSO 4 .7H 2 O, 4.99 g FeSO 4 .7H 2 O, 1.61g CoCl 2 .6H 2 O, 1.57g CuSO 4 .5H 2 O, 1.1g (NH 4 ) 6 Mo 7 o 24 .4H 2 O, 50g Na 2 EDTA, dissolved in water, adjusted to pH 6.5-6.8 with 20% KOH, dilute to 11), dissolved in 975mL w...

Embodiment 2

[0049] Example 2: Transformation of Chinese Tissue Kallikrein Gene

[0050] According to the known Chinese tissue kallikrein gene (GenBank accession number: AY703451) and the preferred codon table of Chlamydomonas reinhardtii (http: / / www.chlamy.org / ), respectively design and artificial synthesis suitable for Chlamydomonas reinhardtii The cKKn gene for nuclear expression, the cKKch1 gene for Chlamydomonas reinhardtii chloroplast expression and the cKKmt gene for Chlamydomonas reinhardtii mitochondrial expression. At the same time, RBCS2 intron 1 (GneBank accession number: X04472) was inserted into the cKKn gene replaced by preferential codons, and the cKKit1 gene containing RBCS2 intron 1 was synthesized by artificial synthesis. The cKKn gene sequence is shown in SEQ ID NO.1, the cKKit1 gene sequence is shown in SEQ ID NO.2, the cKKch1 gene sequence is shown in SEQ ID NO.3, and the cKKmt gene sequence is shown in SEQ ID NO.4.

[0051] The genes cKKn, cKKit1, cKKch1 and cKKmt w...

Embodiment 3

[0052] Example 3: Construction of hK1 expression gene Chlamydomonas expression vector

[0053] (1) Construction of hk1 expression gene Chlamydomonas reinhardtii nuclear expression vector

[0054] Plasmid pckkn18 contains cKKn gene, which can be obtained by Nhe I and Pmac I double digestion. Plasmid pckkit118 contains cKKit1 gene, which can be obtained by NheI and PmacI double digestion. The vector pH105 (Wu JX et al. Efficient expression of green fluorescent protein (GFP) mediated by a chimeric promoter in Chlamydomonas reinhardtii. Chinese Journal of Oceanology and Limnology. Accepted) was cloned with HSP70A-RBCS2 promoter sequence and RBCS2 terminator sequence, the middle Carrying multiple cloning restriction sites such as PmacI, NheI, XbaI and SalI, foreign genes can be inserted and expressed in the nuclear genome of Chlamydomonas reinhardtii. Vector pSP124 (Lambreras v, StevensDR, Purton S. Efficient foreign gene expression in Chlamydomonas reinhardtii mediated by an end...

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Abstract

The invention is a constructing method of a genetic modified chlamydomonas reinhardtii to express kallikrein of human tissue. Firstly kallikrein gene sequence suitable for expression of chlamydomonas reinhardtii kernel genome or expression of chloroplast genome or expression of mitochondrial is designed according to kallikrein gene sequence of human tissue and preferred codon table of chlamydomonas reinhardtii, the gene sequence is synthesized by artificial synthesis, kallikrein gene of human tissue containing non-coded sequence is artificially synthesized, then expression vector of chlamydomonas reinhardtii kernel genome or expression vector of chlamydomonas reinhardtii chloroplast genome or expression vetor of chlamydomonas reinhardtii mitochondrial with mention foreign gene is constructed, then transformed into chlamydomonas reinhardtii by genetic transformation, and genetic modified algal strains capable of expressing kallikrein of human tissue is obtained after screening. The invention lays foundation of treating and preventing some diseases such as hypertention, glomerulonephritis with human original kallikrein gene chlamydomonas reinhardtii and products there.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a method for constructing a transgenic Chlamydomonas reinhardtii expressing human tissue kallikrein. Background technique [0002] With the development of social economy, the improvement of people's living standards, and the acceleration of the pace of work, the pressure on urban residents is increasing. The latest survey results from countries such as the United Kingdom, the United States, and Sweden show that by 2025, 1.56 billion people in the world will suffer from high blood pressure. In 2004, a survey report by the State Council of my country showed that the prevalence rate of hypertension among adults in my country was 18.8%. It is estimated that 160 million people in the country suffer from hypertension, and more than 3 million people are added every year. Hypertension is a common cardiovascular disease and an "invisible killer" that destroys the heart, brain, and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/57C12N15/79C12N1/13C12R1/89
Inventor 胡章立吴锦霞
Owner 深圳市微宇生物科技有限公司
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