假型病毒顆粒

欲以共同轉染三個質體──含有gag-pol gene的pMD.gagpol質體、含有 β-galactosidase gene的pBAG質體以及含有病毒外膜蛋白的表現質體（如：

pcDNA3-E14、pcDNA3-D3E、pAmpho gp85）到 293T 細胞株中，希望可以產生出一帶有登革病毒外膜蛋白之小鼠白血病病毒（MLV）的假型病毒顆粒【圖二十六】。首先先確認這三個質體都可以正常表現：pMD.gagpol 質體由Northern blot結果可以確定在 293T細胞株中有表現gag-pol gene的 mRNA；pBAG質體轉染到 293T細胞後可用X-gal staining使細胞呈藍色；

pcDNA3-E14 與pcDNA3-D3E質體由Northern blot結果可以確定在 293T細胞株中有表現E gene的mRNA，接著進行共同轉染實驗，結果由圖二十五的數據顯示出對照組跟實驗組的blue cell數量差距不大，理論上對照組pAmpho gp85 所產生的blue cell應至少有 10³ CFU/ml以上(Lavillette et al., 2002;

Blaise et al., 2004)，可是在經過幾次重複實驗後，卻一直無法有效提高假型病毒顆粒的產率，推測可能的原因之一為共同轉染的效率不佳，以X-gal staining檢測共同轉染後的細胞，約有四至五成的細胞可以呈現藍色，若以 pBAG五成左右的表現率當作細胞轉染效率，而推測pMD.gagpol及pAmpho gp85 的轉染效率也大概如此，則三個質體共同轉染到同一個細胞內的機率大概只有一成，而這一成的細胞才可能可以產生出假型病毒顆粒，因而造成對照組pAmpho gp85 的假型病毒顆粒產率一直無法提高。

為了解決此共同轉染效率不高的問題，我嘗試從三個質體共同轉染到細胞減為只共同轉染兩個質體到BHK-21 或 293T 細胞，而第三個質體則改由利用登革病毒直接感染共同轉染的細胞以提供外膜蛋白，希望能提高產率，但是由圖二十六的數據顯示無假型病毒顆粒的產生，推測可能原因其一為共同轉染效率依舊不高；其二為由Nothern blot 結果【圖二十五】顯示 pMD.gagpol 在 BHK-21 的表現量極為稀少到偵測不到或是 pMD.gagpol 在 BHK-21 細胞中根本不會表現，若是如此，那當然是不可能會有假型病毒顆

粒的生成了。但在293T 之中也一樣沒有結果，因此尚有其他因素。

在嘗試了以上的實驗後，仍無法成功的產生假型病毒顆粒，究其原因，

推測除了質體轉染效率不高之外，共同轉染之質體之間的比例不正確可能也是原因之一，例如病毒的外膜蛋白若表現過多對細胞可能也會造成傷害，

因此可試著將表現病毒外膜蛋白的表現質體比例降低。又其他因素如細胞的健康狀況是否良好等也都可能是影響結果不理想的原因，所以未來可繼續改善方法與技術以成功產生假型病毒顆粒。

綜合上述的實驗結果得知：(一)登革病毒二型的全長外膜蛋白(DV-2 full-length E protein)在 E.coli Novablue(DE3)之表現，可使用 anti-His-HRP 抗 體藉由Western blot 偵測到 recombinant protein 的表現，惟在哺乳類 BHK21 細胞株中無法偵測到此蛋白。(二)由穩定細胞株的細胞融合現象推測 E protein 會表現在細胞膜上。(三) 雖然在我目前的實驗條件下，還無法成功的產生假型病毒顆粒，但根據前人研究證明：小鼠白血病病毒（MLV）與 C 型肝炎病毒（HCV）組合成假型病毒顆粒是可行的(Bartosch et al., 2003)，

而 C 型肝炎病毒屬於黃質病毒科，其與登革病毒有相同的結構及組成，所以未來可繼續改善方法與技術以成功產生假型病毒顆粒。

伍、結論

疾病管制局www.cdc.gov.tw

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Structure (Camb) 12: 1607-18.

XhoⅠ

E14-sequence (1) CTCGAGGACAATGCGTTGCATAGGAATATCAAATAGAGACTTTGTAGAAG PL046-E (1) ---ATGCGTTGCATAGGAATATCAAATAGAGACTTTGTAGAAG M29095-E (1) ---ATGCGTTGCATAGGAATATCAAATAGAGACTTTGTAGAAG

E14-sequence (51) GGGTTTCAGGAGGAAGCTGGGTTGACATAGTCTTAGAACATGGAAGCTGT PL046-E (41) GGGTTTCAGGAGGAAGCTGGGTTGACATAGTCTTAGAACATGGAAGCTGT M29095-E (41) GGGTTTCAGGAGGAAGCTGGGTTGACATAGTCTTAGAACATGGAAGCTGT

E14-sequence (101) GTGACGACGATGGCAAAAAACAAACCAACATTGGATTTTGAACTGATAAA PL046-E (91) GTGACGACGATGGCAAAAAACAAACCAACATTGGATTTTGAACTGATAAA M29095-E (91) GTGACGACGATGGCAAAAAACAAACCAACATTGGATTTTGAACTGATAGA

E14-sequence (151) AACAGAAGCCAAACAATCTGCCGCTCTAAGGAAGTACTGTATAGAGGCAA PL046-E (141) AACAGAAGCCAAACAATCTGCCGCTCTAAGGAAGTACTGTATAGAGGCAA M29095-E (141) AACAGAAGCCAAACAACCTGCCACTCTAAGGAAGTACTGTATAGAGGCAA

E14-sequence (201) AGCTGACCAACACAACAACAGAATCTCGCTGCCCAACACAAGGAGAACCC PL046-E (191) AGCTGACCAACACAACAACAGAATCTCGCTGCCCAACACAAGGAGAACCC M29095-E (191) AGCTGACCAACACAACAACAGATTCTCGCTGCCCAACACAAGGAGAACCC

E14-sequence (251) AGCCTAAATGAAGAGCAGGACAAAAGGTTCGTCTGCAAACACTCCATGGT PL046-E (241) AGCCTAAATGAAGAGCAGGACAAAAGGTTCGTCTGCAAACACTCCATGGT M29095-E (241) AGCCTAAATGAAGAGCAGGACAAAAGGTTCGTCTGCAAACACTCCATGGT

E14-sequence (301) GGACAGAGGATGGGGAAATGGATGTGGATTATTTGGAAAAGGAGGCATTG PL046-E (291) GGACAGAGGATGGGGAAATGGATGTGGATTATTTGGAAAAGGAGGCATTG M29095-E (291) GGACAGAGGATGGGGAAATGGATGTGGACTATTTGGAAAAGGAGGCATTG

E14-sequence (351) TGACCCGTGCTATGTTCACATGCAAAAAGAACATGAAAGGAAAAGTCGTG PL046-E (341) TGACCCGTGCTATGTTCACATGCAAAAAGAACATGAAAGGAAAAGTCGTG M29095-E (341) TGACCTGTGCTATGTTCACATGCAAAAAGAACATGAAAGGAAAAGTCGTG

E14-sequence (401) CAACCAGAAAACTTGGAATACACCATTGTGATAACACCTCACTCAGGGGA PL046-E (391) CAACCAGAAAACTTGGAATACACCATTGTGATAACACCTCACTCAGGGGA M29095-E (391) CAACCAGAAAACTTGGAATACACCATTGTGATAACACCTCACTCAGGGGA

E14-sequence (451) AGAGCATGCAGTCGGAAATGACACAGGAAAACATGGCAAGGAAATCAAAA PL046-E (441) AGAGCATGCAGTCGGAAATGACACAGGAAAACATGGCAAGGAAATCAAAA M29095-E (441) AGAGCATGCAGTCGGAAATGACACAGGAAAACATGGCAAGGAAATCAAAA

E14-sequence (501) TAACACCACAGAGTTCCATCACAGAAGCAGAGTTGACAGGCTATGGCACT PL046-E (491) TAACACCACAGAGTTCCATCACAGAAGCAGAGTTGACAGGCTATGGCACT M29095-E (491) TAACACCACAGAGTTCCATCACAGAAGCAGAGTTGACAGGCTATGGCACT

E14-sequence (551) GTCACGATGGAGTGCTCTCCGAGAACGGGCCTCGACTTCAATGAGATGGT PL046-E (541) GTCACGATGGAGTGCTCTCCGAGAACGGGCCTCGACTTCAATGAGATGGT M29095-E (541) GTCACGATGGAGTGCTCTCCGAGAACGGGCCTCGACTTCAATGAGATGGT

E14-sequence (601) GTTGCTGCAAATGGAAAATAAAGCTTGGCTGGTGCACAGGCAATGGTTCC PL046-E (591) GTTGCTGCAAATGGAAAATAAAGCTTGGCTGGTGCACAGGCAATGGTTCC M29095-E (591) GTTGCTGCAAATGGAAAATAAAGCTTGGCTGGTGCACAGGCAATGGTTCC

E14-sequence (651) TAGACCTGCCGTTGCCATGGCTGCCCGGAGCGGACACACAAGGATCAAAT PL046-E (641) TAGACCTGCCGTTGCCATGGCTGCCCGGAGCGGACACACAAGGATCAAAT M29095-E (641) TAGACCTGCCGTTGCCATGGCTGCCCGGAGCGGACACACAAGGATCAAAT

E14-sequence (701) TGGATACAGAAAGAGACATTGGTCACTTTCAAAAATCCCCATGCGAAGAA PL046-E (691) TGGATACAGAAAGAGACATTGGTCACTTTCAAAAATCCCCATGCGAAGAA M29095-E (691) TGGATACAGAAAGAGACATTGGTCACTTTCAAAAATCCCCATGCGAAGAA

E14-sequence (751) ACAGGATGTTGTTGTTTTGGGATCCCAAGAAGGGGCCATGCACACAGCAC PL046-E (741) ACAGGATGTTGTTGTTTTGGGATCCCAAGAAGGGGCCATGCACACAGCAC M29095-E (741) ACAGGATGTTGTTGTTTTGGGATCCCAAGAAGGGGCCATGCACACAGCAC

E14-sequence 801) TCACAGGGGCCACAGAAATCCAGATGTCATCAGGAAACTTACTGTTCACA PL046-E (791) TCACAGGGGCCACAGAAATCCAGATGTCATCAGGAAACTTACTGTTCACA M29095-E (791) TCACAGGGGCCACAGAAATCCAGATGTCATCAGGAAACTTACTGTTCACA

E14-sequence (851) GGACATCTCAAGTGCAGGCTGAGGATGGACAAACTACAGCTCAAAGGAAT PL046-E (841) GGACATCTCAAGTGCAGGCTGAGGATGGACAAACTACAGCTCAAAGGAAT M29095-E (841) GGACATCTCAAGTGCAGGCTGAGGATGGACAAACTACAGCTCAAAGGAAT

E14-sequence (901) GTCATACTCTATGTGCACAGGAAAGTTTAAAGTTGTGAAGGAAATAGCAG PL046-E (891) GTCATACTCTATGTGCACAGGAAAGTTTAAAGTTGTGAAGGAAATAGCAG M29095-E (891) GTCATACTCTATGTGCACAGGAAAGTTTAAAGTTGTGAAGGAAATAGCAG

E14-sequence (951) AAACACAACATGGAACAATAGTTATCAGAGTACAATATGAAGGGGACGGT PL046-E (941) AAACACAACATGGAACAATAGTTATCAGAGTACAATATGAAGGGGACGGT M29095-E (941) AAACACAACATGGAACAATAGTTATCAGAGTACAATATGAAGGGGACGGT

E14-sequence (1001) TCTCCATGTAAGATCCCTTTTGAGATAATGGATTTGGAAAAAAGACATGT PL046-E (991) TCTCCATGTAAGATCCCTTTTGAGATAATGGATTTGGAAAAAAGACATGT M29095-E (991) TCTCCATGTAAGATCCCTTTTGAGATAATGGATTTGGAAAAAAGACATGT

E14-sequence (1051) TTTAGGTCGCCTGATTACAGTCAACCCAATCGTAACAGAAAAAGATAGCC PL046-E (1041) TTTAGGTCGCCTGATTACAGTCAACCCAATCGTAACAGAAAAAGATAGCC M29095-E (1041) TTTAGGTCGCCTGATTACAGTCAACCCAATCGTAACAGAAAAAGATAGCC

E14-sequence (1101) CAGTCAACATAGAAGCAGAACCTCCATTCGGAGACAGCTACATCATCATA PL046-E (1091) CAGTCAACATAGAAGCAGAACCTCCATTCGGAGACAGCTACATCATCATA M29095-E (1091) CAGTCAACATAGAAGCAGAACCTCCATTCGGAGACAGCTACATCATCATA

E14-sequence (1151) GGAGTAGAGCCGGGACAATTGAAGCTCAACTGGTTTAAGAAAGGAAGTTC PL046-E (1141) GGAGTAGAGCCGGGACAATTGAAGCTCAACTGGTTTAAGAAAGGAAGTTC M29095-E (1141) GGAGTAGAGCCGGGACAATTGAAGCTCAACTGGTTTAAGAAAGGAAGTTC

E14-sequence (1201) TATCGGCCAAATGCTTGAGACAACAATGAGGGGAGCGAAGAGAATGGCCA PL046-E (1191) TATCGGCCAAATGCTTGAGACAACAATGAGGGGAGCGAAGAGAATGGCCA M29095-E (1191) TATCGGCCAAATGATTGAGACAACAATGAGGGGAGCGAAGAGAATGGCCA

E14-sequence (1251) TTTTAGGTGACACAGCTTGGGATTTTGGATCCCTGGGAGGAGTGTTTACA PL046-E (1241) TTTTAGGTGACACAGCTTGGGATTTTGGATCCCTGGGAGGAGTGTTTACA M29095-E (1241) TTTTAGGTGACACAGCTTGGGATTTTGGATCCCTGGGAGGAGTGTTTACA

E14-sequence (1301) TCTATAGGAAAGGCTCTCCACCAAGTTTTCGGAGCAATCTATGGGGCTGC PL046-E (1291) TCTATAGGAAAGGCTCTCCACCAAGTTTTCGGAGCAATCTATGGGGCTGC M29095-E (1291) TCTATAGGAAAGGCTCTCCACCAAGTTTTCGGAGCAATCTATGGGGCTGC

E14-sequence (1351) CTTCAGTGGGGTCTCATGGACTATGAAAATCCTCATAGGAGTCATTATCA PL046-E (1341) CTTCAGTGGGGTCTCATGGACTATGAAAATCCTCATAGGAGTCATTATCA M29095-E (1341) CTTCAGTGGGGTCTCATGGATTATGAAAATCCTCATAGGAGTCATTATCA

E14-sequence (1401) CATGGATAGGAATGAATTCACGCAGCACCCCACTGTCTGTGTCACTAGTA PL046-E (1391) CATGGATAGGAATGAATTCACGCAGCACCCCACTGTCTGTGTCACTAGTA M29095-E (1391) CATGGATAGGAATGAATTCACGCAGCACCTCACTGTCTGTGTCACTAGTA

E14-sequence (1451) TTGGTGGGAGTCGTGACGCTGTATTTGGGAGTTATGGTGCAGGCCTAGTC PL046-E (1441) TTGGTGGGAGTCGTGACGCTGTATTTGGGAGTTATGGTGCAGGCC--- M29095-E (1441) TTGGTGGGAGTCGTGACGCTGTATTTGGGAGTTATGGTGCAGGCC---

XbaⅠ E14-sequence (1501) TAGAGGGCCC PL046-E (1486) --- M29095-E (1486) ---

圖一、定序 pcDNA3-E14 質體上之 insert （DV-2 E gene）序列與 Taiwan local strain PL046 及 New Guinea-C（M29095）作序列比對。相異處以標 示之。此核酸序列包含轉譯起始密碼與終止密碼（以方框□標示）。

EcoRV NotI XhoI XbaI

pET-30a(+)-E14

XbaI enzyme digestion

XbaI NotI XhoI XbaI ↓ ↓↓ ↓

His-tag D2E full-length

TAA

以實驗室已有之cDNA clones 為模板（template）

PCR with primer (E14His-F+E14His-R)

Sac I Xho I

圖四、pET-30a(+)△5T-E14 質體建構之示意圖 D2E full-length

clone into pET-30a(+)△5T SacI、Xho I

pET-30a(+)∆5T-E14 6754 bp

Kanamycin

T7 promoter His-tag

D2E ( E14 ) XhoI (159)

SacI (1654)

pET-30a(+)△5T-E14

以實驗室已有之cDNA clones 為模板（template）

PCR with primer (D3E-F+D3E-R)

Bam HI Xba I

pcDNA3-D3E

6858 bp

D3E(full-length) P CMV

T7 promoter Ampicillin

Neomycin

BamHI (908)

XbaI (2396)

TAA D3E full-length

clone into pcDNA3 Bam HI、Xba I

圖五、pcDNA3-D3E 質體建構之示意圖

(A) pcDNA3-E14 質體；Lane3 為 AflⅢ切割 pcDNA3-E14 質體。A~H 為酵素 切割pcDNA3-E14 質體後所得之片段：A 約 5.4kb；B 約 1.5kb；C 約 5.6kb；

D 約 0.8kb；E 約 0.5kb；F 約 3.1kb；G 約 2.0kb；H 約 1.8kb。M:size marker。

(A) pET-30a(+)-E14 質體；Lane 3 為 BamHI 切割 pET-30a(+)-E14 質體。A~G 為酵素切割pET-30a(+)-E14 質體後所得之片段：A 約 5.5kb；B 約 1.4kb；

C 約 5.4kb；D 約 1.5kb；E 約 5.6kb；F 約 0.8kb；G 約 0.5kb。M:size marker。

(A)

圖八、利用限制酵素確認 pcDNA3-His-E14 質體 (A) pcDNA3-His-E14 之 map。

(B) Lane 1 為 HindⅢ切割 pcDNA3-His-E14 質體；Lane2 為 BamHI 切割 pcDNA3-His-E14 質體；Lane3 為 AflⅢ、XbaI 切割 pcDNA3-His-E14 質 體。A~L 為酵素切割 pcDNA3-His-E14 質體後所得之片段：A 約 6.2kb；

B 約 0.6kb；C 約 0.3kb；D 約 5.6kb；E 約 0.8kb；F 約 0.5kb；G 約 0.3kb；

H 約 2.6kb；I 約 2.0kb；J 約 1.3kb；K 約 0.7kb；L 約 0.4kb。M:size marker。

pET-30a(+)∆5T-E14 (A) pET-30a(+)△5T-E14 之 map。

(B) Lane 1 為 SacI、Xho I 切割 pET-30a(+)△5T-E14 質體；Lane2 為 Bam HI 切割pET-30a(+)△5T-E14 質體；Lane3 為 NcoI、AvaI 切割 pET-30a(+)△

5T-E14 質體； Lane4 為 AflⅢ切割 pET-30a(+)△5T-E14 質體。A~L 為酵 素切割pcDNA3-D3E 質體後所得之片段：A 約 5.3kb；B 約 1.5kb；C 約 5.5kb；D 約 0.8kb；E 約 0.5kb；F 約 4.3kb；G 約 1.2kb；H 約 0.8kb；I 約0.4kb；J 約 2.7kb；K 約 2.1kb；L 約 1.9kb。M:size marker。

(A) I 切割 pcDNA3-D3E 質體；Lane3 為 Nco I 切割 pcDNA3-D3E 質體。A~L 為酵素切割pcDNA3-D3E 質體後所得之片段：A 約 5.4kb；B 約 1.5kb；

C 約 2.3kb；D 約 2.0kb；E 約 1.6kb；F 約 0.7kb；G 約 0.2kb；H 約 3.3kb；

I 約 1.8kb；J 約 0.7kb；K 約 0.5kb；L 約 0.4kb。M:size marker。

M 1 2 3 4 (pcDNA3-tE-HAHis)在 E.coli BL21(DE3)的蛋白質表現

(A) 12% SDS-PAGE 以 Coomassie blue 染色之結果。(B)以 anti-His-HRP 抗體偵測蛋白質表現之 Western blot 的結果。(C)以 anti-HA-HRP 抗體偵測蛋白質表現之Western blot 的結果。M 為 size marker；lane 1 為 pcDNA3 質體轉形至E.coli BL21(DE3)之 total protein 的上清液；lane 2 為 pKRY1 質體轉 形至E.coli BL21(DE3) 之 total protein 的上清液；Lane 3 為 pcDNA3 質體轉 形至E.coli BL21(DE3) 之 total protein 的 pellet；lane 4 為 pKRY1 質體轉形 至E.coli BL21(DE3) 之 total protein 的 pellet。

1 2 3 4 5 6 M

圖十二、以 anti-DV-2 domainⅢ抗體偵測 pKRY1(pcDNA3-tE-HAHis)及 pcDNA3-E14 在 E.coli BL21(DE3)的蛋白質表現

(A) 12% SDS-PAGE 以 Coomassie blue 染色之結果。(B)以 anti-DV-2 domain

Ⅲ抗體偵測蛋白質表現之Western blot 的結果。M 為 size marker；lane 1 為 pcDNA3 質體轉形至 E.coli BL21(DE3) 之 total protein 的上清液；lane 2 為 pKRY1 質體轉形至 E.coli BL21(DE3) 之 total protein 的上清液；lane 3 為 pcDNA3-E14 質體轉形至 E.coli BL21(DE3) 之 total protein 的上清液；Lane 4 為 pcDNA3 質體轉形至 E.coli BL21(DE3) 之 total protein 的 pellet；lane 5 為 pKRY1 質體轉形至 E.coli BL21(DE3) 之 total protein 的 pellet；lane 6 為 pcDNA3-E14 質體轉形至 E.coli BL21(DE3) 之 total protein 的 pellet。三角 記號標示為truncated E protein 表現之位置。

nt 1855 nt 1730 nt 1666 nt 182 ↓ ↓↓ ↓

ATG His-tag thrombin S-tag enterokinase D2E full-length

TAA ～7 kDa ～55 kDa

圖十三、pET-30a(+)-E14 質體轉譯蛋白質之示意圖

170 kDa－

圖十四、以 anti-His-HRP 抗體偵測 pET-30a(+)-E14 在 E.coli Novablue(DE3) 的蛋白質表現

(A) 12% SDS-PAGE 以 Coomassie blue 染色之結果。(B) 以 anti-His-HRP 抗體偵測蛋白質表現之 Western blot 的結果。M 為 size marker；lane 1 為 pET-30a(+)質體轉形至 E.coli Novablue(DE3) 之 total protein 的上清液；lane 2 為 pET-30a(+)-E14 質體轉形至 E.coli Novablue(DE3) 之 total protein 的上 清液；lane 3 為 pET-30a(+)質體轉形至 E.coli Novablue(DE3) 之 total protein 的pellet；Lane 4 為 pET-30a(+)-E14 質體轉形至 E.coli Novablue(DE3) 之 total protein 的 pellet。

170 kDa－

圖十五、以 anti-His-HRP 抗體偵測 pcDNA3-His-E14 在 E.coli Novablue (DE3)的蛋白質表現

(A) 10% SDS-PAGE 以 Coomassie blue 染色之結果。(B) 以 anti-His-HRP 抗體偵測蛋白質表現之Western blot 的結果。M 為 size marker；lane 1 為 pcDNA3 質體轉形至 E.coli Novablue(DE3) 之 total protein 的上清液；lane 2 為 pcDNA3 質體轉形至 E.coli Novablue(DE3) 之 total protein 的 pellet；lane 3 為pcDNA3-His-E14 質體轉形至 E.coli Novablue(DE3) 之 total protein 的上青 液；Lane 4 為 pcDNA3-His-E14 質體轉形至 E.coli Novablue(DE3) 之 total protein 的 pellet。

nt 1678 nt1648 nt 157 nt 140 ↓ ↓ ↓ ↓

ATG D2E full-length His-tag

TAA ～55 kDa ～0.7 kDa

圖十六、pET-30a(+)△5T-E14 質體轉譯蛋白質之示意圖

170 kDa－

圖十七、以 anti-His-HRP 抗體偵測 pET-30a(+)△5T-E14 在 E.coli Novablue (DE3)的蛋白質表現

(A) 10% SDS-PAGE 以 Coomassie blue 染色之結果。(B) 以 anti-His-HRP 抗體偵測蛋白質表現之 Western blot 的結果。M 為 size marker；lane 1 為 pET-30a(+)△5T 質體轉形至 E.coli Novablue(DE3) 之 total protein 的上清 液；lane 2 為 pET-30a(+)△5T 質體轉形至 E.coli Novablue(DE3) 之 total protein 的 pellet；lane 3 為 pET-30a(+)△5T-E14 質體轉形至 E.coli Novablue

在文檔中表現登革病毒2型PL046外膜蛋白以用於建構假型小鼠白血病病毒 (頁 55-108)

伍、結 論

陸、參考文獻

6858 bp

伍、結論