Codon Optimization Of Human Insulin Gene And The Application Of An Endogenous Promoter From Tremella Fuciformis

In this study, Tremella fuciformis wild strain Tr 21-01 and pBHg-BCA was used as test strain and test plasmid. The main process is as follows:The results of the T. fuciformis codon usage bias analysis were served as references to optimize and synthesize the codons of human insulin gene BCA in vitro, and the optimized BCA was named as BCA1. The promoter gpd was replaced with p-G06. Under the guidance of two strategies codon optimization and promoter replacement, three eukaryotic expression vectors were built by combining traditional vector construction method and advanced In-Fusion method. Three newly constructed plasmids were transformed into the spore of Tr 21-01 by efficient agrobacterium-mediated transformation (AMT) method. PCR, RT-PCR and ELISA were applied for screening out T. fuciformis positive transformants that could express human insulin. The content of BCA or BCA1 gene in the genome of each positive transformant relative to that of BCA gene in the genome of Tr 26 was achieved from relative quantification in real-time qRT-PCR. Detailed results were shown as follows:1) Human insulin gene was codon optimized and synthesized. With reference to the results of the T. fuciformis codon usage bias analysis and without changing the amino acid sequence of the expressed product,29 codons of BCA gene were replaced. BCA then became BCA1, which was in accordance with the preferrance of T. fuciformis. What’s more, multiple clone sites and protected nucleosides were added to the both ends of BCA1, i.e.259 bp DNA fragment MCS1-BCA1-MCS2. After synthesized, this DNA fragment was inverted into the plasmid pMD18-T and became a new plasmid named pUC-BCA1(1).2) Construction of eukaryotic expression vectors. Combining advanced In-Fusion clone method, traditional vector-constructed method was used to construct vectors. After the construction of two intermediate vectors pUC-BCA1(2) and pBHg-BCA1(2), three eukaryotic expression vectors pBHg-BCA1-G06, pBHg-BCA-G06 and pBHg-BCAl-gpd were achieved and verified by sequencing.3) AMT method was used in the genetic transformation of T. fuciformis. Mediated by Agrobacterium EHA105, three vectors pBHg-BCA-G06, pBHg-BCAl-gpd and pBHg-BCAl-G06 were transformed into the spore of Tr 21-01 by AMT method and a number of putative transformants obtained. After twice screening by hygromycin and cefotaxime,33 putative transformants of each vectors were randomly picked out for conservation, respectively named Tr 301-333, Tr 401-433 and Tr 501-533.4) The screening of T. fuciformis positive transformants and identification of highly expressed strains. PCR, RT-PCR and ELISA were used to screen the 99 putative transformants of three vectors pBHg-BCA-G06, pBHg-BCAl-gpd and pBHg-BCA1-G06,17,5 and 5 T. fuciformis positive transformants that can express human insulin were achieved. Codon optimization and promoter replacement applied alone can improve the expression level of human insulin gene by 38.9% and 50.2%, can improve the percentage of highly expressed strains by 40% and 58%. In contrast, the effect on the expression of human insulin genes comes from the latter were more significant than that from the former. However, the combined application can improve the expression level and the percentage by 104.5% and 100%. So the combined effect was more significant than the separate one comes from the two strategies.5) qRT-PCR was used to detect the Ct values of foreign gene BCA or BCA1 and internal reference gene GPD of the genome of all the above 27 T. fuciformis positive transformants, and using 2-△△Ct algorithm to calculate the content of BCA or BCA1 gene of each transformants relative to that of BCA of Tr 26. As the results showed, the contents of BCA or BCA1 of all the T. fuciformis transformants’genomes are greatly different from each other; there was no significant correlation between the content of BCA or BCA1 and the expression level of human insulin; promoter replacement had a highly significant inhibitory effect on the inverting of BCA or BCA1 into the genome of T. fuciformis, while codon optimization played a significant role in promoting.