CN1382734A

CN1382734A - Process for preparing human insuline from transgenic romaine lettuce

Info

Publication number: CN1382734A
Application number: CN 02104176
Authority: CN
Inventors: 林忠平; 倪挺; 胡鸢雷; 高音
Original assignee: Individual
Current assignee: Individual
Priority date: 2002-03-15
Filing date: 2002-03-15
Publication date: 2002-12-04

Abstract

A process for preparing human insulin from transgenic romaine lettuce as a bioreactor which can be also used as a food to prevent and cure diabetes is disclosed. The human insulin gene is artificially synthesized through designing codon, synthesizing several DNA fragments, splicing them together and adding KDEL endoplasmic reticulum retention sequence to its terminal C. Under the drive of CaMV35S promoter, the said gene is transferred into ramaine lattuce and the human insulin is expressed in the leaves and stems of ramaine lattuce.

Description

Utilize transgenic lettuce production insulin human's method

The present invention utilizes romaine lettuce to come manufacturer's Regular Insulin as a kind of plant bioreactor.This insulin human can enter in the body by mucocutaneous membrane on the digestive tube as a kind of oral preparations by eating romaine lettuce raw, improves the autoimmunity situation of diabetic.Also can extract the insulin human of high expression level amount simultaneously, and obtain the injection insulin human from this romaine lettuce.

The point of main innovation of the present invention is that (1) select the codon of plant-preference for use, the synthetic human insulin gene; (2) add KDEL endoplasmic reticulum retention sequence at synthetic insulin human's C end, avoided the degraded of Regular Insulin in vegetable cell.

The research that utilizes potato tuber to produce Hepatitis B virus vaccine in plant bioreactor research has been subjected to paying attention to widely.Romaine lettuce is produced oral polypeptide drug with it and is had very big application potential as eating vegetables or fruit.The insulin human is crucial for the urine patient's of insulin-dependent treatment.Some diabetic subject's metabolism adjusting is relevant with the autoimmunity function.It is effective (Reddy S et at, Pancreas2000,20 (1): 55-60 that oral insulin has been proved to be sick treatment of the urine of some type; Ploix C et at, Diabetes 1999,48 (1) 2150-6; Ramiya VK, Maclaren NK, Horm Res 1997,48 suppl 4:67-70).

One of technical essential of this invention is the synthetic of insulin gene.Why not adopting natural human insulin's dna sequence dna, is because we adopt the codon of plant-preference can improve the expression amount of this gene in plant.The encoded amino-acid sequence of natural insulin A chain and B chain of our used human insulin gene, but the DNA sequence of DNA sequence and natural human insulin's gene and inequality.

Two of the main points of this invention are structures of plant expression vector pCAM35S-InsK.Be connected on the p11Z carrier human insulin gene InsK of synthetic and order-checking, downcut the CaMV 35S promoter from the pBI221 carrier then, place the upstream of insulin gene.CaMV 35S-InsK downcut be placed on before the no terminator of plant expression vector pCAMBIA1390, cut with PCR assay certificate vector construction correct through enzyme.

Three of the main points of this invention are the endoplasmic reticulum retention sequences that added KDEL at the C of human insulin gene end.Studies have shown that four aminoacid sequences that add KDEL at proteinic C end, this protein is stranded in the endoplasmic reticulum, thereby avoids being degraded in a large number.

Four of the main points of this invention are that agriculture bacillus mediated romaine lettuce transforms.Change plant expression vector pCAM35S-InsK freeze-thaw method over to Agrobacterium LBA4404.Agrobacterium breeds the back and cultivates altogether with the romaine lettuce blade in containing the YEB substratum of Streptomycin sulphate, the blade of cultivating callus occurs after placing for some time on the screening culture medium that contains Cb and Totomycin altogether, the callus differentiation grows seedling then, seedling is moved into respectively in the different bottles, identify positive plant through PCR, and further Southern blotting and ELISA evaluation.

In one embodiment of the invention, about synthesizing of human insulin gene, select the codon of plant-preference for use.In 63 bases of insulin human A chain, 13 bases have been changed for this reason.In 90 bases of B chain, change 18 bases, added 12 alkali yl coding endoplasmic reticulum retention sequence KDEL at 3 ' end in addition.We have designed 6 amino acid whose junction fragments between A chain and B chain.The principle of design of the amino acid kind of this junction fragment and order is to keep the natural Three Dimensions Structure of insulin human.We clone EcoRI and BamHI site in the pGEM-11Z carrier with comprise the insulin human A chain and B chain and the middle segment order of connection of synthetic, and are determined this plasmid called after p11Z-InsK through the analysis and the sequential analysis of plasmid enzyme restriction collection of illustrative plates.In this embodiment, the human insulin gene fragment can obtain by pcr amplification, during amplification, the primer of use is as follows: InsK 5 ': 5 '-GAT CCA TGT TCG TTA ACC AAC ACC-3 ' InsK 3 ': 5 '-CTC GTC CTT GTT GCA GTA GTT CTC-3 '.

In one embodiment of the invention, human insulin gene places under the regulation and control of composition type expression promoter CaMV35S promotor, changes in the intermediate carrier again.Further change binary expression vector again over to, finish the structure of a plant expression vector and (see the clone of human insulin gene of Fig. 1, synthetic and the structure schema of plant expression vector.Wherein human insulin gene is under the control of constitutive promoter CaMV35S).

The bacterial classification that this patent relates to is in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, Zhong Guan-cun, unit address BeiJing, China, this microorganism classification name is called colon bacillus (Ecoli.), the preservation code name is p11Z-InsK, deposit number is CGMCC NO.0723, and preservation date is on March 8th, 2002.One, synthetic human insulin gene InsK sequence: 1 GATCCATGTT CGTTAACCAA CACCTTTGCG GATCTCACCT TGTTGAGGCT CTTTACCTTG61 TTTGCGGAGA GAGGGGATTC TTCTACACCC CAAAGACCGG AGGACCATCT GGAGGAGGAA121 TCGTTGAGCA ATGCTGCACC TCTATCTGCT CTCTTTACCA ACTTGAGAAC TACTGCAACA181 AGGACGAGCT TTAAG

Claims

1, utilizes transgenic lettuce production recombinant human insulin's method.The gene that wherein is used to transform comprises that also acceptor also comprises other plants such as cucumber, Radix Dauci Sativae, tobacco except that romaine lettuce through insulin gene and the partial sequence of transforming thereof.

2, according to the method for claim 1.Used gene also comprises not through the insulin gene of transforming and part is transformed except through the transformed insulin gene of plant-preference cipher word.

3, according to the method for claim 1.Used gene comprises also that except 3 ' end has added the insulin gene of KDEL sequence 3 ' end does not add the gene of KDEL sequence and the gene that has added other like.

4, according to the method for claim 1.Used gene also comprises the method that adopts other amino acid to connect except adopting the method for 6 amino acid with INSULIN A, two chains connections of B.

5, according to the method for claim 1.The promotor that insulin gene adopts except the CaMV 35S promoter, also comprise other can be in plant expression promoter.

6, according to the method for claim 1.Acceptor also comprises other plants such as cucumber, Radix Dauci Sativae, tobacco except that romaine lettuce.