| The chloroplast genetic engineering is one of the effective ways to express foreign proteins. Compared with the nucleus transformation, it has several advantages: such as the big gene copy number, the high expression rate, the stable inheritable character, the high biological safety, avoiding of gene silencing and position effect, etc. Additionaly, the translation efficiency is a key point for determining the expression ability. Some translation enhancer sequence can improve the expression of the recombinant proteins obviously. It has been demonstrated that a length of nine bases (UUAACUUUA) which originate from bacteriophage T7gene10leader sequence can increase the expression quantity of foreign genes by40-340-fold in the Escherichia coli. The principle may be that it can strengthen the combining capacity of mRNA and the16S ribosome to promote translation.To make the chloroplast transformation come true, an effective transformation vector is needed. This kind of transformation vector must have several basic elements: a homologous recombination arm, an integrated expression cassette of foreign gene (including promoter, target gene and regulatory sequence) and a selectable marker. Lux Ct is transformed properly based on the luciferase gene Lux AB. It has an efficient endogenous atpA gene promoter,5’UTR and3’UTR. Franklin et al used the cassette of atpA promoter-atpA5’UTR-gfp gene-rbcL3’UTR to express the green fluorescent protein successfully in the chloroplast of Chlamydomonas reinhardtii. Lux Ct is one kind of shuttle vectors, it can express in the prokaryotic organism, as well as in the eukaryotic organism, but it can not be induced to express.The growth and metastasis of tumors are a process which depended on angiogenesis. Canstatin is a new kind of endogenous Angiostatin which was found in recent years. It is the non-collagenous1(NCI)domain of a2chain of type IV collagen. In vitro, it can restrain the hyperplasia and migration of vascular endothelial cells, thus leading to the apoptosis of the endothelial cell. In vivo, it can inhibit the growth of tumors effectively. At present, this kind of dormancy therapy through the suppression of angiogenesis has a broader application prospect in clinical trials. In our research, we constructed the human canstatin expression vector to validate the fuction of the g10-L translation enhancer sequence through detecting the change of expression quantity of the canstatin protein.MethodsWe designed three pairs of primers according to the cDNA sequence of human canstatin on Genbank at first. The total RNA extracted from the non-small-cell lung carcinoma was used as a template and then the RT-PCR method was used to clone the total length of human canstatin gene and the cDNA fragments of human canstatin which have g10-L translation enhancer sequence (UUAACUUUA) in the downstream of the5’UTR and in the upstream of the3’UTR. Subsequently, the fragments were cloned into the pMD18-T vector and identified by digestion of restriction enzymes, respectively. Through the method of digestion and connection of restriction enzymes, a human canstatin chloroplast expression vector with the cassette of atpA5’UTR-canstatin gene-rbcL3’UTR from the cassette of atpA5’UTR-gfp gene-rbcL3’UTR was constructed. Simultaneously, human canstatin chloroplast expression vectors were constructed which had the g10-L translation enhancer sequence (UUAACUUUA) in the downstream of the5’UTR and in the upstream of the3’UTR, respectively. The recombinant expression vectors were identified by digestion of restriction enzymes PaeR7Ⅰ and Sph Ⅰ. In order to add the g10-L translation enhancer sequence in the upstream of the5’UTR and in the downstream of the3’UTR,we used the constructed recombination vector with the cassette of atpA5’UTR-canstatin gene-rbcL3’UTR as the template, to design two pairs of primers for PCR. We obtatined the fragments of atpA5’UTR-canstatin gene-rbcL3’UTR with the g10-L translation enhancer sequence in the upstream of the5’UTR and in the downstream of the3’UTR, respectively. The fragments were cloned into the pMD18-T vector and identified by digestion of restriction enzymes Ava I and Sph I, respectively. The prokaryotic expression vectors with the g10-L translation enhancer sequence in the upstream of the5’UTR and in the downstream of the3’UTR were constructed respectively. At last, the constructed expressin vectors were expressed in the Escherichia coli DH5a, respectively. The proteins were extracted and detected by the method of western blotting in order to validate the function of the the g10-L translation enhancer sequence(UUAACUUUA)that it can improve the expression of foreign proteins at a initial step.Results1.The amplification of the human canstatin gene and the human canstatin gene fragments with the g10-L translation enhancer sequence (UUAACUUUA) in the downstream of the5’UTR and in the upstream of the3’UTRThe total RNA extracted from the A549cell using TRIzol reagent was perfect. The two bands of28S and18S were clear. Using RT-PCR, the human canstatin gene was amplified, as well as the human canstatin gene fragments with the g10-L translation enhancer sequence in the downstream of the5’UTR and in the upstream of the3’UTR, which contained about700bp. The fragments were cloned into the pMD18-T vector and were identified by digestion of restriction enzymes, respectively.2.The construction of human canstatin chloroplast expression vector and the human canstatin chloroplast expression vector with the g10-L translation enhancer sequence (UUAACUUUA) in the downstream of the5’UTR and in the upstream of the3’UTRUsing the method of digestion and connection of restriction enzymes, the human canstatin chloroplast expression vectors with the cassette atpA5’UTR-canstatin gene-rbcL3’UTR were constructed, as well as the human canstatin chloroplast expression vectors with the g10-L translation enhancer sequence in the downstream of the5’UTR and in the upstream of the3’UTR, respectively. It showed that the vectors were constructed successfully through the digestion of restriction enzymes.3.The supplementary construction of the human canstatin prokaryotic expression vector with the g10-L translation enhancer sequence (UUAACUUUA)in the upstream of the5’UTR and in the downstream of the3’UTRAccording to the methods above-mentioned, the human canstatin prokaryotic expression vectors with the g10-L translation enhancer sequence in the upstream of the5’UTR and in the downstream of the3’UTR were constructed successfully,which were identified through the digestion of restriction enzymes. The human canstatin prokaryotic expression vectors with the g10-L translation enhancer sequence in the downstream of the5’UTR and in the upstream of the3’UTR were constructed successfully at the same time.4. The expression and detection of the human canstatin protein by western blottingThe constructed vectors were expressed in the Escherichia coli DH5a, respectively. The extracted proteins were detected by western blotting, respectively. The result showed that there was a specific band at24KDa. In addition, the bands of the expression vectors with the translation enhancer sequence (UUAACUUUA)were stronger than the bands of those without the translation enhancer sequence. The results validated the function of the the g10-L translation enhancer sequence that it can improve the expression of foreign proteins.Conclusion1. This study has successfully constructed the human canstatin chloroplast expression vector and the human canstatin chloroplast expression vector with the g10-L translation enhancer sequence in the downstream of the5’UTR and in the upstream of the3’UTR, respectively.2.This study has successfully constructed the human canstatin prokaryotic and the human canstatin prokaryotic expression vector with the g10-L translation enhancer sequence in the upstream and the downstream of the5’UTR and the3’UTR, respectively.3.The result of western blotting indicates that the g10-L translation enhancer sequence can increase the expression of foreign proteins. |
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