| Atrial Natriuretic Peptide (ANP), a small peptide consisting of 28 amino acids, is important involved in fluid and electrolyte balance. The major physiological effects of ANP are vasodilation, natriuresis, and inhibition of the renin-angiotensin-aldosterone (RAA) and the sympathetic nervous systems. ANP is been applied in clinical tratment for heart failure and acute respiratory distress syndrome (ARDS). Although preparation of ANP using genetic engineering should encounter many difficulties for its character of a small peptide, comparing with the complication of chemical synthesis methodology, expression of ANP using gene technology is more advantage in industrial application because of its convenience and efficiencyThe major objective of this study is to obtain human Atrial Natriuretic Peptide (α-hANP) as recombinant drug by the means of genetic engineering and protein engineering. In order to realize this purpose, a series of constructs carrying the gene of hANP were made and transformed into Escherichia.coli. system system for over-expression. Through fermentation, high yield of hANP was obtained stably. Protein purification and large scale production of genetic engineering drug were future explored. At the same time, bi-functional peptide with molecular targeting function and new ANP derivatives were designed. A novel strategy of construction of "three peptides from mono-strain" was also developed. In addition, increasing target gene expression was achieved by the new technique of tandem tandem-joined operons, which improves the yield of desired protein in E.coli system by.way of increasing the dose of target gene.The thesis comprises three sections:First section touches the genetic engineering research on human a-Atrial Natriuretic Peptide ANP (α-hANP). Stable E.coli strain with high-yield of human ANP was first established. Recombinantα-hANP was then purified and standardized. Its pharmacological activities were evaluated. Second section illustrates the investigation of "three peptides from mono-strain" and genetic engineering for ANP derivatives. It consists of the construction, expression, purification and primary pharmacological activities evaluation of bi-functional peptide DFP3 with targeting ability. Besides, ANP derivative sANP2 was constructed and using the similar fusion gene entity of targeting bi-functional peptide mentioned above, to construct DFP2, producing DFP2 fusion protein. Third section examines the feasibility of linking operons in tandem to enhance expression of heterologous genes in E.coli. and clarify the potential control mechanism of the total plasmid DNA amount in each host cell. In the first section, the DNA segment (Ad) encoding the 6 amino acids Gly-Val-Arg-Gly-Pro-Arg of thrombin adapter sequence was fused with ANP gene of 28 peptides using PCR. Later it was cloned into sequencing vector pBSsk to form the plasmid pCW1. for sequencing comfirm. This Ad-ANP sequence was then sub-cloned into expression vector pLY1 on the downstream of PL promoter, becoming expressive cartilage PL-SD-Ref-(α-hANP)-rrnBT1T2. The constructed plasmid pCW111was then transformed into E. coli DH5αstrain and expressed the fusion protein Ref-Ad-ANP under 42℃thermal induction, of fusion protein Ref-ANP expressed by pCW111 was 45％of total cell protein with a molecular weight of 22, 834Da. The product existed as inclusion bodies in E. coli. Yields Tandem repeating of the 2.25kb expressive cartilage PL-SD-Ref-(α-hANP)-rrnBT1T2 was performed to acquire plasmids pCW111, pCW112, pCW113, pCW114 with 1～4 expression cartilages and expression level of fusion protein being 46％, 54.8％, 56％and 60.1％of total cell protein respectively.The designed fusion protein Ref-Ad-ANP from strain DH5α/pCW112 was over-expreessed in shaking flask culture. At the same time high-density fermentation using 10L fermenter was studied. About 100g of engineered E. coli pellet was harvested per liter medium with an expression level of 25％. The engineered E. coli was disrupted and washed to obtain the inclusion bodies. The inclusion bodies was dissolved and purified with reverse phase chromatography to produce the fusion protein Ref-Ad-ANP with a purity of approximately 90％. This is followed by optimizing the thrombin treatment of fusion protein in which the effect was above 90％in 2 M urea. After cleavage reaction, the sample was subjected to gel filtration (size exclusion) chromatography, the purity of the ANP released was above 85％in this step. After polishing by reverse phase chromatography, the final product of ANP could reach a purity of above 95％. The recovery of ANP per liter of shaking flask was no less than 3mg/L. Physiochemical standards of this genetic engineered ANP was identified with the drug production requirement. High-resolution MALD-TOF evaluation revealed the molecular weight of the ANP produced was similar to the theoretical value. Amino acid sequencing of the first 20 N-terminal of ANP product was parallel with the theoretical value. The amino acids composition was similar with standard material. The iso-electric reading of the ANP product was 9.5～10 with capillary isoelectric focusing and its purity being single symmetrical peak compared to standard. High Pressure Liquid Chromatography determined the purity of the final product being 96.7％. The primary pharmacological evaluation showed the ANP produced had the effects of blood pressure lowering in vivo, diuresis in vivo and vasodilation in vitro, which is similar to the activity of standard ANP. Pilot process was then explored for high density fermentation, inclusion bodies preparation, fusion protein and ANP purification. This is a key to commercial production of this genetically engineered h-αANP.In the second phase of research, two mutation points were introduced into interleukin 2 (IL2) gene forming IL2a. Upon sequence conformation, IL2a was fused with Ad-ANP to produce IL-2a-Ad-ANP gene. The product of this gene was DFP3 fusion protein with bi-functional targeting entity, realizing the construction of fusion gene "3 peptides from mono strain". Using the method in phase 1, DFP3 gene was sub-cloned into the downstream of PL promoter, creating expressive cartilage PL-SD-DFP1-T1T2 and into pCW120. PCW120 was capable of producing the target protein with a molecular weight of 19244D as inclusion bodies by thermo-induction of E. coli. However the expression level was under 30％. The distance between SD sequence and DFP3 promoter was adjusted using the sub-cloning procedure, introducing 2 nucleotides TA. This leads to the expression plasmid of pCW121. Using the tandem repeating subcloning to produce higher expression level of DPF3. The plasmids constructed here, pCW121, pCW122, pCW123 and pCW124 have 1～4expression cartilages and their production level of DFP3 were 34％, 44％, 47％and 51％of the total protein, respectively. The inclusion bodies produced by engineered DH5α/pCW122 under shaker culture were washed and purified, later renatured through slow dilution and modified with citric acid anhydride, returning the proper folding of fusion protein DFP3. This fusion protein, upon anion exchange chromatography achieved a purity of around 90％. In vivo diuresis and in vitro vaso-dilation action of DFP3 was similar with the standard ANP while remitting the acute anuria state caused by IL2. After thrombin cleavage, two recombinant products with ANP and IL2 activities respectively, would be released. Primary studies confirmed the possibility and rationality of "three peptides from mono-strain" proposal.Also in the second section, to increase the activity and stability of ANP, high expression vector carrying IL2a-Ad-sANP2 fusion gene was engineered. The yield of this fusion protein reached 39％of total protein. One component released from this fusion product is sANP2, which is a 22-peptide derivative from the original 28-peptide ANP. Compared with the natural one, the first 1～6 amino acids of ANP were deleted in sANP2. In addition, the 8th Phe was mutated to Thr and the 12th Met to lie in sANP2.In the third section of the thesis, these two series of expression constructs, pCW11 and pCW12, were utilized here on the analysis of the effect of tandem repeating cloning on the target protein expression and on the change of plasmid copy. Protein expression was determined via SDS-PAGE and laser-density scanning. Plasmid copy number was measured by the aid of incorporation of 3H-TdR. The results proved that the tandem repeating technique could improve the gene dose and thus improve the expression of target protein in E. coli system. The data also showed that the With increasing size, plasmid copy number decreased, however, target gene dosage increased significantly (P＜0.01). Further calculation showed that the total amount of plasmid DNA per cell are no significant different in each series It's suggested that based on the same culture condition, for specific E. coli strain, the total DNA amount of plasmid in one host cell was constant to some extent. The research of this thesis included all phases of developing genetically engineered ANP recombinant drugs in lab stage. Through the aforesaid lab research, the technique routine of small peptide genetic engineering was newly created, and genetically engineered ANP recombinant drugs and its derivatives were developed, realizing the idea of "3 peptides from mono-strain". Some parts of the research findings had obtained 2 national patents. The lab achievement regarding ANP genetic engineering study has been transferred to and finished the industrial trial and pre-clinical assay for its therapeutic effciency and pharmaco-toxicology. At present, it is being applied for clinical trial.
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