10384 200326107 UDC Cloning and Expression of Genes from Venom Gland of Bungarus multicinctus 2006 7

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V 1 1 2 C 4 α- P22-A31 0 11 1 11 2 11 13 α- 13 2 α- 14 3 α- 15 4 α- 15 17 α cdna 21 1 21 2 23 24 α NGF 24 α 24 3 3 RACE 28 31 5 α- cdna a

6 PCR 35 36 α- cdna 36 α- cdna 36 C 41 45 1 45 2 56 56 C 3 RACE 5 RACE 56 2 C C 58 3 C 60 4 C 60 5 C 62 6 64 7 C 64 8 C 65 9 C 66 67 1 2 C cdna 67 2 C 68 3 C 68 4 C 69 85 87 88 b

Table of Contents Abstract(in Chinese) V Introduction 1 Neurotoxin 4 2 C-type lectin 9 Part I Expression and functional analysis of recombinant α-bungarotoxin (P22-A31) Introduction 0 Materials and Methods 11 1 Materials 11 2 Methods 11 Results 13 Expression and purification of recombinant α-bungarotoxin 13 2 Detection of α-bungarotoxin s immunogenicity 14 3 Toxicity of the recombinant α-bungarotoxin 15 4 Analgesic effects of recombinant α-bungarotoxin 15 17 Part II Cloning of α-bungarotoxin genes and analysis on polymorphism of α-bungarotoxin cdna Introduction Materials and Methods 21 1 Materials 21 2 Methods 23 Results 24 Cloning of α-bungarotoxin and NGF 24 Analysis of α-bungarotoxin genomic sequences 24 3 Sequencing of NGF 3 RACE products 28 a

31 5 Alignment of different α-bungarotoxin cdna sequences 6 Analysis of artificial mutation from PCR process 35 Discussion 36 α-bungarotoxin cdna is not result from different transcrip 36 Analysis of reasons for α-bungarotoxin cdna 36 Part III Cloning and Expression of C-type lectin from Bungarus multicinctus Introduction 41 Materials and Methods 45 1 Materials 45 2 Methods 56 Results 56 Amplification of C-type lectins full length cdna 56 2 Alignment of several C-type lectins from different snakes 58 3 Construction of expression vectors 60 4 Recombinant expression of C-type lectins 60 5 Expression, refolding and purification of recombinant C-type lectins 62 6 Production, purification and detection of antibody for C-type lectins 64 7 Detection of natural C-type lectin in Bungarus multicinctus 64 8 Hemagglutation assay of recombinant C-type lectins 65 9 Purification of natural C-type lectins by antibody 66 Discussion 67 1 Two full length cdnas of C-type lectins 67 2 Expression of recombinant C-type lectins 68 3 Bioactivity of recombinant C-type lectins 68 4 Natural C-type lectins in Bungarus multicinctus 69 Reference 85 Published papers 87 Acknowledgements 88 b

pgex-bgtx P22-A31 BL21 DE3 α- 1.225 mg/l α- α- LD 50 1.598 mg/kg α- 1/5 1/4 LD 50 55.2% 1/8 LD 50 20.5% α- P22-A31 α- α- cdna 5 α- cdna cdna 12 α- cdna α- cdna α- cdna RNA PCR DNA RACE cdna 2 C cdna BML-1 BML-2 BML-2 Bungarus multicinctus 2 135 137 pet-his BL21 DE3 plyss C 135 C 137 C 50% 50% Western blotting C 4 14.4KD C I

C α- C II

Cloning and Expression of Genes from Venom Gland of Bungarus multicinctus ABSTRACT α-bungarotoxin plays very important role in neuroscience research, clinical application and the pharmaceutical industry. In order to acquire quantites of α-bungarotoxin, we expressed GST-α-bungarotoxin fusion protein using constructed plasmid pgex-bgtx (P22-A31) in E. coli BL21 (DE3) cell and finally obtained recombinant α-bungarotoxin with yields of about 1.225 mg/l. The results of both ELISA and Western blot showed that recombinant α-bungarotoxin has the same antigenicity as natural α-bungarotoxin. In vivo toxicity tests showed that the LD 50 of recombinant α-bungarotoxin was 1.598 mg/kg, about 1/5 that of natural α-bungarotoxin. Analgesis percentages with doses of 1/4 LD 50 and 1/8 LD 50 were 55.2% and 20.5% respectively, indicating that recombinant α-bungarotoxin possesses analgesic efficiency. It is disputed whether there is polymorphism in cdna of α-bungarotoxin, and what is the mechanism to result of this phenomenon. In order to further study this question, we cloned and sequenced α-bungarotoxin gene from the same individual and nerve growth factor cdna from the same reverse transcription products by Wang et al, and analyzed their mutation rates. Those results indicate that polymorphism of α-bungarotoxin cdna is not transcripted from genomic DNA or RNA editing, but results from reverse transcription process, PCR, and gene cloning. C-type lectins are found in many animals and bind in a Ca 2+ -dependent fashion to mono- and oligosaccharides. In our research, we cloned two C-type lectin full-length cdna from Bungarus multicinctus venom gland by RACE technology, named BML-1, BML-2. It is the first report to clone BML-2 cdna from venom gland of Bungarus multicinctus. We also constructed expression vector using 135 and 137 amino acids of two C-type lectins ligated into pet-his plasmid and then expressed III

then in BL21 DE3 plyss cell. Recombinant protein is inclusion body and account for 30% full protein of expression bacteria and the refolding rate is 30%. The refolded recombinant C-type lectins are able to agglutinate rabbit erythrocytes, Recombinant BML-1, BML-2 with 135 amino acids are the monomers, and recombinant BML-1, BML-2 with 137 amino acids are dimmers of 50%, monomers of 50%. Western blotting and purification of natural C-type lectins showed that C-type lectins form dimers in physiological condition. Cloning and expression of C-type lectins from Bungarus multicinctus venom is very important to further study characteristics, structure and functions of C-type lectins in Bungarus multicinctus. Keywords: venom protein, α-bungarotoxin, C-type lectin, cloning, recombinant expression IV

10 15 12 18 85% 90% 90% 1 6,000 12,000 ph 9 20% 50% 60 62 4 [1] 70 74 5 [2] κ- 66 5 [3 4] κ- [5 9] 4 (presynaptically-acting neurotoxin) β- β-neurotoxin [10] (postsynaptically-acting neurotoxin) α- α-neurotoxin [11] (ion-channel neurotoxin) (anticholinesterase neurotoxin) Elapidae Hydrophiidae α- N- 80 α- 60 62 4 66 74 5 2 3 β β- 1

12 N- α- α- 60-62 4 α- 5 3 α- α- [12] 20%~50% α- 74 1 α- a 185-196 2 1. α- [13] FIG 1. Crystal structure of α-bungarotoxin. 2. α- a 185-196 [13] FIG 2. NMR Structure of α-bungarotoxin Free and Bound to a Mimotope of the Nicotinic Acetylcholine Receptor. 3. - [14] FIG 3. Crystal structure of -bungarotoxin. Aird [15] - N 1 1 RGD 2

- α- κ- 3 κ- α- κ- Arg-34 Pro-36 α- κ- cdna 3 α- κ- 3 2 [16] FIG 4. 4. Gene organization of long neurotoxin. FDA 2 3

Cobroxin Nyloxin 2 C A 2 C C κ unitz-type C [17] 2.1 C C 115 130 αβ CRD [18] C [19] [19, 20] I versican, aggrecan, neurocan brevican [21], C C II C asialo III C collectins C SP-A SP-D conglutinin C IV selectin, 3 selectins L-selectin E-selectin 4

P-selectin V C II C II [22] IgE [23] VI C I 1 fibronectin II CRD C 1 DEC-205 VII C CRD [17] 2.2 C Russel s viper (Vipera russeli) X RVV-X [24] C RVV-X C C IX/X [25] IX [26] Agkistrodon acutus X [27, 28] botrocetin VWF Ib [29, 30] bothrojaracin, Bothrops jararaca hirudin α [31] 80 C C [17] C C CRD VII C C C A B 2.3 C C Crotalus atrox [32], B. jararaca [33], 5

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