Rcr gene sequence with plant favored codon

A technique for favoring codons and gene sequences, applied in the field of Rcr gene sequences

Inactive Publication Date: 2006-07-26
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] After searching the prior art documents, it was found that although Fu Yong et al. (Tumor Research and Clinic, 2004, 16 (4): 217-220) successfully expressed Rcr using the prokaryotic system (Escherichia coli), but bacteria including bacteria Compared with some eukaryotic expression systems such as yeast, animal cells, transgenic animals and higher plant expression systems, prokaryotic expression systems have obvious defects in terms of safety, production costs and large-scale production (Fischer, et al.Transgenic Research, 2000 , 9:279-299), but there is no report on the successful production of Rcr in plants by designing and synthesizing the Rcr gene according to plant-biased codons

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Rcr Gene Synthesis with Sequence Information and Homology Analysis

[0035] 1. Rcr gene synthesis (gene synthesis)

[0036] The Rcr gene was designed by our laboratory according to the principle of plant preferred codons and commissioned by Shanghai Sangong to synthesize it and then ligated into the pUC18 vector, namely pUC18-Rcr;

[0037] 2. Rcr gene sequence information

[0038] The full length of the Rcr gene synthesized according to plant preference codon design and synthesis of the present invention is 463bp (including the linker consisting of protective bases and restriction site sequences), and the detailed sequence is shown in SEQ ID NO.1, wherein the open reading frame is located at 51-449 bit nucleotides. The deduced amino acid sequence of the full-length Rcr has a total of 133 amino acid residues, a molecular weight of Mw=14762.2, and an isoelectric point of pi=9.23. See SEQ ID NO.2 for the detailed sequence.

[0039] 3. Rcr gene homology analysis

[004...

Embodiment 2

[0072] Rcr gene expression vector construction

[0073] 1. Restriction enzyme cutting

[0074] The pUC18 cloning vector containing the Rcr gene (pUC18-Rcr) was digested with Bam H I and Sac I to obtain a DNA fragment of the Rcr gene with Bam H I and Sac I sticky ends (the same as the expression vector) (the gene can be designed with BamH I and Sac I sites);

[0075] 2. Connection (ligation)

[0076] Connect the excised gene Rcr into the plasmid pBI121 that has been excised with Bam H I and Sac I or the large fragment of the pCAMBIA2300 plant expression vector transformed with pBI121, and detect the transformed Escherichia coli by blue and white screening or PCR to obtain positive results. clone.

[0077] 3. Gene PCR detection

[0078] According to the nucleotide sequence of the gene, design primers: Rcr F: 5'-atg tgc gct aag tctctt ctt c-3' (forward primer), Rcr R: 5'-tgg gca tct tcc aat tcc agc-3 '(reverse primer) is to carry out PCR amplification using the vector containi...

Embodiment 3

[0081] Eukaryotic Expression of Rcr Gene in Tobacco and Tomato

[0082] 1. Preparation of engineering bacteria containing Rcr gene expression vector

[0083] The plant expression vector containing the Rcr gene obtained in Example 2 was identified or sequenced by enzyme digestion, and under the premise of ensuring that the reading frame of the target gene (Rcr gene) in the expression vector was correct, the constructed expression vector was then frozen Transformation of Agrobacterium (such as EHA105) into engineering bacteria for plant genetic transformation, transformation of tomato and tobacco by Agrobacterium-mediated method.

[0084] 2. Agrobacterium-mediated transformation of tomato

[0085] ① Sterilize the seeds of tomato (Lycopersicon esculentum.var.cerasiforme cv.Yellow fruit No.22) (22# yellow cherry tomato) with 75% ethanol and 20% sodium hypochlorite and sow them on 1 / 2 MS medium, and wait for 6-8 days After the leaves are unfolded, the cotyledons or hypocotyls are...

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PUM

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Abstract

Disclosed is a Rcr gene sequence with plant favored codon and synthesized DNA molecule which comprises, nucleotide sequence for encoding the polypeptides having Rana catesbeina Rcr protein reactivity, the nucleotide sequence has at least 70% homology with the position No.51-449 nucleotide sequence in the nucleic acid SEQ ID NO.1, the sequence encodes polypeptide of the amino acid sequence represented by SEQ ID NO.2, the invention can be applied to the preparation of medicament for treating early stage tumor transition.

Description

technical field [0001] The present invention relates to a gene sequence in the field of biotechnology, more specifically, relates to a Rcr gene sequence according to plant preferred codons. Background technique [0002] Bullfrog ribonuclease (Rana catesbeiana RNase, Rcr) has a small molecular weight and strong cytotoxicity, and is a commonly used cytotoxin in the clinical treatment of malignant tumors. At present, Rcr for clinical use is mainly obtained through tissue extraction. Due to the limitation of tissue source and Rcr content, the clinical application of Rcr is limited (Liao, et al. Protein Expr Purif. 1996, 7(2): 194-202). With the development and application of molecular biology and genetic engineering technology, it has become possible to produce Rcr by genetic engineering technology, which provides a new way of thinking for the production of Rcr and the alleviation or solution of the shortage of clinical supply. [0003] After searching the prior art documents, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N9/22
Inventor 赵凌侠崔丽洁唐克轩开国银钱虹妹陈玉辉张慧
Owner SHANGHAI JIAO TONG UNIV
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