魏宇雙,劉姣揚,韓瑩倩,郭珍珍,劉曉賀,巴 根,韓立強,王 江,褚貝貝,楊國宇(河南農(nóng)業(yè)大學農(nóng)業(yè)部動物生化與營養(yǎng)重點開放實驗室,鄭州 450002)
豬繁殖與呼吸綜合征(porcine reproductive and respiratory syndrome, PRRS)是一種以豬繁殖與呼吸綜合征病毒(porcine reproductive and respiratory syndrome virus, PRRSV)為病原體的疾病,可造成仔豬和育成豬呼吸障礙、母豬流產(chǎn),具有較高的致死率,給全世界養(yǎng)豬產(chǎn)業(yè)造成巨大經(jīng)濟損失[1-3]。目前關于PRRSV的研究多集中于病毒學特性、病毒的起源、進化以及豬體對其產(chǎn)生的免疫應答等方面[4-5],而關于PRRSV如何依賴宿主因子完成吸附、入侵、RNA脫殼、核酸蛋白質(zhì)等生物大分子合成、子代病毒組裝和釋放生活周期的分子機制的研究尚不充分。
組織蛋白酶 (cathepsin) 是在各種動物組織的細胞內(nèi)(特別是溶酶體部分)發(fā)現(xiàn)的一類蛋白酶,是半胱氨酸蛋白酶家族的主要成員,在生物界已發(fā)現(xiàn)20余種,人體中主要存在11種,是近年來備受關注的一類靶標蛋白酶[6-7]。研究表明cathepsin與多種病理過程相關,例如cathepsin G通過切割白細胞介素6受體、水解活化細胞因子等途徑參與血管性炎癥、急性肺損傷等炎癥性疾病的發(fā)生[8];由于cathepsin D的表達水平與胃癌的淋巴結(jié)轉(zhuǎn)移之間的相關性,其表達水平的變化可以作為胃癌預后的判斷指標之一[9]。而cathepsin在病毒復制周期中的作用也陸續(xù)被發(fā)現(xiàn),例如埃博拉病毒(Ebola virus, EBOV)入侵宿主細胞時,需要借助cathepsin B和cathepsin L使病毒表面糖蛋白構(gòu)象改變從而使病毒可以將遺傳物質(zhì)“注入”宿主細胞[10-11];在肝癌細胞中,乙肝病毒的x基因 (HBx) 表達量與cathepsin S的表達量正相關,在瞬時過表達HBx的HepG2細胞中,HBx可能是通過上調(diào)cathepsin S的表達量促進細胞增殖[12];PRRSV通過激活NF-κB 通路和cathepsin L來上調(diào)乙酰肝素酶的表達,降解硫酸乙酰肝素從而促進病毒的釋放[13]。由此,筆者推測其它組織蛋白酶也可能參與PRRSV的生活周期。本研究構(gòu)建了針對猴源cathepsin基因的shRNA真核慢病毒表達載體,與包裝質(zhì)粒 (pMD2.G, psPAX) 共同轉(zhuǎn)染 HEK293T/17細胞后收取慢病毒上清液,感染源于MA-104的非洲綠猴胚胎腎上皮細胞Marc145細胞系(對PRRSV易感),獲得高效且穩(wěn)定敲減cathepsin基因的細胞株。該穩(wěn)定細胞系的建立為后續(xù)探究cathepsin在PRRSV生活周期中的作用奠定了基礎。
pLKO.1載體、慢病毒包裝載體psPAX2、pMD2.G、Ampicillin和Puromycin購自Sigma公司;人胚胎腎細胞HEK293T/17購自ATCC公司;猴胚胎腎上皮細胞Marc145來自河南省動物免疫學重點實驗室;PRRSV-GFP毒株由西北農(nóng)林科技大學周恩民教授饋贈;DMEM、FBS以及胰酶購自Gibco;Prime Script RT Reagent Kit、SYBR Premix ExTaqⅡ、rTaq酶、DL10000 DNA Marker、DL2000 DNA Marker購自TaKaRa公司;限制性內(nèi)切酶EcoRⅠ-HF、AgeⅠ-HF及T4連接酶購自NEB公司;Lipofectamine?3000 Transfection Kit購自Promega公司;質(zhì)粒中提試劑盒為QIAGEN產(chǎn)品; TOP10大腸桿菌感受態(tài)為實驗室自制。引物由上海生工生物工程有限公司合成;DNA測序由上海英濰捷基生物公司完成。
1.2.1 猴源cathepsin基因shRNA重組質(zhì)粒的構(gòu)建和鑒定 RNA干擾 (RNA interference, RNAi) 是一種進化上保守的抵御外來病毒或轉(zhuǎn)基因的防御機制[14-15]。將與靶基因mRNA同源的雙鏈RNA (double strand RNA, dsRNA) 導入細胞,shRNA的發(fā)卡結(jié)構(gòu)可被細胞切割成siRNA,然后siRNA 結(jié)合到RNA 誘導沉默復合物上(RNA-induced silencing complex,RISC),該復合物能夠結(jié)合到目的mRNA上并將其特異性地降解,從而產(chǎn)生相應的功能表型缺失[16-19]。本研究利用慢病毒載體把shRNA導入細胞,其在感染后可以整合到細胞的基因組上;并且載體中的U6 啟動子確保了shRNA 長時間表達;這種整合的shRNA 序列可隨細胞DNA一起復制并被傳遞到子代細胞中去,從而使靶基因長期穩(wěn)定沉默。
首先,查找NCBI數(shù)據(jù)庫得到非洲綠猴(Chlorocebus sabaeus)的全部cathepsin基因;然后,使用Invitrogen公司在線軟件BLOCK-iT RNAi Designer,針對每種猴源cathepsin基因設計3條長度21 bp的shRNA序列,每條序列作用于不同靶點。shRNA退火形成雙鏈,與pLKO.1載體(經(jīng)EcoRⅠ-HF、AgeⅠ-HF處理)連接成重組質(zhì)粒(如圖1);轉(zhuǎn)化大腸桿菌,37 ℃振蕩培養(yǎng)1 h,涂布于含100 μg·mL-1Ampicillin 的固體 LB 培養(yǎng)基倒置培養(yǎng)過夜。次日挑選陽性單克隆,以菌液為模板,PCR 擴增鑒定插入片段。菌液PCR上游引物:5′-TCGACGGTATCGATCACGAGACTAG-3′;下游引物:5′-AATTGTGGATGAATACTGCCATTTG-3′。陽性結(jié)果經(jīng)上海英濰捷基生物公司測序正確后,按照中提試劑盒說明書抽提質(zhì)粒,置于-20 ℃保存?zhèn)溆谩?/p>
圖1 shRNA表達原理Fig.1 Illustration of shRNA
1.2.2 慢病毒顆粒包裝 將HEK293T/17細胞以1.6×106·瓶-1的密度接種于T25細胞培養(yǎng)瓶,培養(yǎng)至細胞融合度40%左右,按照Lipofectamine?3000 Transfection Kit說明書將重組質(zhì)粒與包裝質(zhì)粒psPAX2、pMD2.G共轉(zhuǎn)染。分別于48、72 h收取上清中的慢病毒,分裝凍存于-80 ℃。
1.2.3 穩(wěn)定轉(zhuǎn)染Marc145細胞系建立 以2×105·孔-1的密度接種Marc145細胞于6孔板,培養(yǎng)至細胞融合度30%~40%,棄去1 mL 培養(yǎng)基,加入1 mL慢病毒感染細胞。48 h 后用含10 μg·mL-1puromycin的DMEM完全培養(yǎng)基持續(xù)篩選至對照組細胞全部死亡。繼續(xù)使用含2 μg·mL-1puromycin的DMEM完全培養(yǎng)基篩選維持,待感染效果鑒定。
1.2.4 實時熒光定量PCR檢測 Trizol法抽提細胞總RNA,反轉(zhuǎn)錄得到cDNA;以實時熒光定量PCR方法測定cathepsinmRNA 表達量的變化,得到敲減效率最高的細胞株。實時熒光定量PCR 引物見表1。
表1非洲綠猴cathepsin基因?qū)崟r熒光定量PCR引物序列
Table1Sequenceofquantitativereal-timePCRprimerforChlorocebussabaeuscathepsingenes
引物名稱Primername序列(5′→3′)Sequence上游引物Forwardprimer下游引物ReverseprimercathepsinACCCTGTGGTGCTTTGGCGGCGACCTCAGTGTCATTAGTTGcathepsinBCAGTGTCCCACCATCAAAGGCCATTACAGCCGTCCCcathepsinCGCGGCTTCCCATACCTCCCTCCATAGAAACCTCCCcathepsinDTGTGGAGGACCTGATTGCCGCTGGACTTGTCGCTGTTGTATcathepsinEGAATACTTCGGCACTATCTCCCACGGTCAGTCCTTCCACcathepsinFGAAGGCCAAGGTCTACATCATGCCATAGCCCACAAGCcathepsinGCCTGGTGCGAGACAACTTTGGATGGTCCGCTGATTcathepsinHACATACAGCACGGAGGAGTGGAAGGTGGGTAGGGAcathepsinKTTGCTGCTGAAACGAACATCCACCTTGCTGTTATcathepsinLATGAAGAAGGATGGAGGAGAAAGCCCAACAAGAACCcathepsinOCCAGCAGGTCATTGACTGTCAAAGGGCCAAAGGTAAGAcathepsinSCGGATTCTGTGGACTGGCGTGGCTTTGTAGGGATAcathepsinVGGGAATACAGCCAAGGGGCACCAGTCGCACTAAAAcathepsinWCCGCCTGGACATCTTTGTGGCTCTTCGGACCCTATcathepsinZAGGGAGAAGATGATGGCAGAACCTCGATGGCAAGGTTGTAT
1.2.5 PRRSV-GFP感染cathepsin shRNA-Marc145細胞株 將篩選得到cathepsin shRNA-Marc145細胞株以1×105·孔-1的密度接種于12孔板,培養(yǎng)至細胞融合度80%。感染PRRSV-GFP病毒 (MOI=1),37 ℃吸附1 h后,用1%FBS的DMEM培養(yǎng)基培養(yǎng)48 h,置于倒置熒光顯微鏡觀察,并用酶標儀檢測熒光結(jié)果。
所構(gòu)建cathepsin基因shRNA 文庫包含15種猴源cathepsin基因,針對每種cathepsin基因分別設計的3個shRNA 靶點序列如表2。
表2猴源cathepsin基因shRNA靶序列
Table2TargetingsequenceofshRNAforChlorocebussabaeuscathepsingenes
目的基因Primername靶點(5′→3′)Targets靶點?1Target?1靶點?2Target?2靶點?3Target?3cathepsinAGGAGTACAAGAACAACAAACTGCACCTCCACTACTGGTTTGTGCTGCTTAGCTCACAGAAATAcathepsinBGGACAGGATCACTGTGGAATCGGATCACTGTGGAATCGAATCGGATCTGCATCCACACCAATGcathepsinCGCATCAAGGTGACCATTTACTGCGAGAATCCGTATACTAACCGGAACGGATGAGTGTGCAATTcathepsinDGCACGGACTCCAAGTATTACAGCCGCTACTACACTGTGTTTGGCAAATACAACAGCGACAAGTcathepsinEGGAGCTTGTCTGGGATCATTGGCTACGACCACTCCCATTTCTGCCCTTCCGACAAGATTAAGCcathepsinFGCCTGTCCGTCTTTGTCAATAGCGAGAAGGGTTACTACTACTGGGTTACTACTACTTGCATCGcathepsinGGGATTCCTCCAGAAGTCTTCAGGAGAACAATGAGAAGCTTCAGGACCATCCAGAATGACATCAcathepsinHGGAAGATAAATGCCCACAACAGCAACTGGAGGAAGATAAATGGCAAGGATGGTGATTGCAAGTcathepsinKGGAAGCAATATAACAGCAAGGGCAGTAATGACACCCTTTATAGCAACAAGCACTGGATAATTAcathepsinLGGGAAACACAGCTTCACAATGGCAGGTGATGAATGGCTTTCAGCCAGACTGTAGCAGTGAAGAcathepsinOGGGAGGCATTATACAGCATCAGCTCTAGTGGAGAAGCAAATCGGTCATTGACTGTTCGTATACcathepsinSGGTGTCTACTATGAACCATCCGGGAGACATGACCAGTGAAGAGGATATATTCGGATGGCAAGAcathepsinVGGGAAACATGGCTTCACAATGGGGAATACAGCCAAGGGAAACGCCATTCGTCCTTCCAGTTCTcathepsinWGGAGCAATACCTGTGGCATCAGCATCAATTTCTTGGATTTCGGCACAGAATTGCCCAGTACCTcathepsinZGCAACAGAAAGACTGGCTAACGGAGAAGATGATGGCAGAAATGGATCAACATCAAGAGGAAGG
用限制性內(nèi)切酶EcoRⅠ-HF和AgeⅠ-HF對pLKO.1 載體進行雙酶切,經(jīng)1%瓊脂糖凝膠電泳分離出7 500 bp 左右的目的條帶(如圖2),回收該條帶得到pLK0.1酶切產(chǎn)物。猴源cathepsin基因shRNA 文庫共包含45對shRNA 寡核苷酸鏈退火雜交后形成雙鏈DNA,亞克隆至pLKO.1得到45個pLKO.1-cathepsinshRNA 重組質(zhì)粒。該重組質(zhì)粒轉(zhuǎn)化至大腸桿菌TOP10 感受態(tài),挑取陽性單克隆,菌液PCR鑒定得到273 bp產(chǎn)物的陽性菌(圖3),測序。
M. DL10000相對分子質(zhì)量標準;1.陰性對照;2. pLKO.1質(zhì)粒雙酶切產(chǎn)物M. DL10000 marker; 1. Negative control; 2. The double-restriction endonuclease digestion products of pLKO.1 plasmids
圖2 pLKO.1質(zhì)粒的雙酶切Fig.2 Restriction enzymes digestion of pLKO.1 plasmids
M. DL2000相對分子質(zhì)量標準(DL2000 marker);1. cathepsin A-1 shRNA-pLKO.1; 2. cathepsin A-2 shRNA-pLKO.1; 3. cathepsin O-1 shRNA-pLKO.1; 4. cathepsin O-2 shRNA-pLKO.1; 5. cathepsin D-1 shRNA-pLKO.1; 6.cathepsin D-2 shRNA-pLKO.1; 7.cathepsin Z-1 shRNA-pLKO.1; 8. cathepsin Z-2 shRNA-pLKO.1; 9.cathepsin B-1 shRNA-pLKO.1; 10. cathepsin B-2 shRNA-pLKO.1; 11. cathepsin C-1 shRNA-pLKO.1; 12. cathepsin C-2 shRNA-pLKO.1; 13. cathepsin E-1 shRNA-pLKO.1; 14. cathepsin E-2 shRNA-pLKO.1; 15. cathepsin F-1 shRNA-pLKO.1; 16. cathepsin F-2 shRNA-pLKO.1; 17. cathepsin G-1 shRNA-pLKO.1; 18. cathepsin G-2 shRNA-pLKO.1; 19. cathepsin H-1 shRNA-pLKO.1; 20. cathepsin H-2 shRNA-pLKO.1; 21. cathepsin L-1 shRNA-pLKO.1; 22. cathepsin L-2 shRNA-pLKO.1; 23. cathepsin K-1 shRNA-pLKO.1; 24. cathepsin K-2 shRNA-pLKO.1; 25. cathepsin S-1 shRNA-pLKO.1; 26. cathepsin S-2 shRNA-pLKO.1; 27. cathepsin V-1 shRNA-pLKO.1; 28. cathepsin V-2 shRNA-pLKO.1; 29. cathepsin W-1 shRNA-pLKO.1; 30. cathepsin W-2 shRNA-pLKO.1; 31. cathepsin A-3 shRNA-pLKO.1; 32. cathepsin B-3 shRNA-pLKO.1; 33. cathepsin C-3 shRNA-pLKO.1; 34. cathepsin D-3 shRNA-pLKO.1; 35. cathepsin E-3 shRNA-pLKO.1; 36. cathepsin F-3 shRNA-pLKO.1; 37. cathepsin G-3 shRNA-pLKO.1;38. cathepsin H-3 shRNA-pLKO.1; 39. cathepsin L-3 shRNA-pLKO.1; 40. cathepsin K-3 shRNA-pLKO.1; 41. cathepsin O-3 shRNA-pLKO.1; 42. cathepsin S-3 shRNA-pLKO.1; 43. cathepsin V-3 shRNA-pLKO.1; 44. cathepsin W-3 shRNA-pLKO.1; 45. cathepsin Z-3 shRNA-pLKO.1
圖3 重組質(zhì)粒的PCR鑒定Fig.3 PCR results of recombinant plasmid (portion)
慢病毒感染Marc145細胞48 h,用含10 μg·mL-1puromycin的DMEM完全培養(yǎng)液培養(yǎng)細胞至對照組細胞死亡,換含2 μg·mL-1puromycin DMEM完全培養(yǎng)基培養(yǎng)維持。抽提對照細胞和敲減細胞全部RNA反轉(zhuǎn)錄得到cDNA,實時熒光定量PCR分析cathepsinmRNA表達量。以β-actin作內(nèi)參,比較對照細胞與不同靶點敲減cathepsin基因的細胞mRNA表達量(如圖4),選出敲減效率最高的細胞株(圖5),各cathepsinshRNA-Marc145細胞株中cathepsinmRNA的表達量均明顯下降,表明成功構(gòu)建cathepsin基因敲減細胞系。為證明該細胞系的穩(wěn)定性,選取同一細胞株不同代次細胞進行敲減效率檢測(圖6),發(fā)現(xiàn)不同代次細胞敲減效率穩(wěn)定。進一步證明所構(gòu)建細胞系高效且穩(wěn)定敲減cathepsin基因。
與shControl相比,**.P<0.001,***.P<0.000 1Compared with shControl, **.P<0.001,***.P<0.000 1
圖4 cathepsin基因不同靶點的敲減效率比較Fig.4 Comparison of knockdown efficiency of cathepsin genes at different targets
與shControl相比,**.P<0.001,***.P<0.000 1Compared with shControl, **.P<0.001,***.P<0.000 1
圖5 cathepsin shRNA-Marc145細胞株相應cathepsin mRNA相對表達量Fig.5 The relative mRNA expression of cathepsin in Marc145 cell lines
shControl. 對照; shCTS A P3. cathepsin A shRNA-Marc145細胞株第3代細胞; shCTS A P8. cathepsin A shRNA-Marc145細胞株第8代細胞; shCTS A P12. cathepsin A shRNA-Marc145細胞株第12代細胞; shCTS D P3. cathepsin D shRNA-Marc145細胞株第3代細胞; shCTS D P9. cathepsin D shRNA-Marc145細胞株第9代細胞: shCTS D P22. cathepsin D shRNA-Marc145細胞株第22代細胞; shCTS V P3. cathepsin V shRNA-Marc145細胞株第3代細胞; shCTS V P10. cathepsin V shRNA-Marc145細胞株第10代細胞; shCTS V P18. cathepsin V shRNA-Marc145細胞株第18代細胞,與shControl相比,**.P<0.001, ***.P<0.000 1shControl. Control; shCTS A P3. The 3rd generation of cathepsin A shRNA-Marc145 cell line; shCTS A P8. The 8th generation of cathepsin A shRNA-Marc145 cell line; shCTS A P12. The 12th generation of cathepsin A shRNA-Marc145 cell line; shCTS D P3. The 3rd generation of cathepsin D shRNA-Marc145 cell line; shCTS D P9. The 9th generation of cathepsin D shRNA-Marc145 cell line: shCTS D P22. The 22nd generation of cathepsin D shRNA-Marc145 cell line; shCTS V P3. The 3rd generation of cathepsin V shRNA-Marc145 cell line; shCTS V P10. The 10th generation of cathepsin V shRNA-Marc145 cell line; shCTS V P18. The 18th generation of cathepsin V shRNA-Marc145 cell line.Compared with shControl, **.P<0.001, ***.P<0.000 1
圖6 不同代次cathepsin shRNA-Marc145細胞株相應cathepsin mRNA相對表達量(部分)Fig.6 The relative mRNA expression of cathepsin in different generations of Marc145 cell lines (partial)
將篩選得到的cathepsinshRNA-Marc145細胞接種于12孔板,培養(yǎng)至細胞融合度80%,按上述方法用PRRSV-GFP分別感染shControl-Marc145細胞和cathepsinL shRNA-Marc145細胞,48 h后置于熒光顯微鏡觀察,發(fā)現(xiàn)cathepsinL shRNA-Marc145細胞中PRRSV熒光強度明顯下降(圖7B),酶標儀檢測結(jié)果與熒光結(jié)果一致(圖7A),表明敲減cathepsinL會顯著抑制PRRSV-GFP的增殖。
非洲綠猴體內(nèi)可以表達cathepsin A、B、C、D、E、F、G、H、K、L、O、S、V、W、Z,本研究設計了針對這些cathepsin基因的shRNA引物(表2),構(gòu)建得到了45個重組質(zhì)粒,命名為pLKO.1-cathepsin shRNA(圖2、圖3)。利用該重組質(zhì)粒包裝慢病毒顆粒并感染Marc145細胞,篩選鑒定出45株穩(wěn)定且高效敲減cathepsin的細胞株,命名為cathepsin shRNA-Marc145細胞株(圖4~6)。這些細胞系經(jīng)PRRSV-GFP病毒感染,初步篩選出對PRRSV復制有影響的cathepsin(圖7)。Guo等[13]的研究表明PRRSV感染誘導乙酰肝素和cathepsin L表達,調(diào)節(jié)乙酰肝素轉(zhuǎn)運到細胞表面降解HS,從而促進PRRSV子代病毒的釋放,這說明cathepsin L在PRRSV的釋放中發(fā)揮著重要作用。筆者的試驗也證實了這一點,如圖7所示,敲減cathepsinL會顯著抑制PRRSV的增殖。因其他有效果cathepsin的分子機制正在進一步解析中,所以在本文中沒有顯示。
PRRSV是高度變異的單股正鏈RNA囊膜病毒,其毒株的多樣加劇了藍耳病的臨床復雜性[20-21]。因此,單純針對其囊膜抗原研制的疫苗很難滿足生產(chǎn)上的防控需求[22-23]。研究顯示,PRRSV復制周期的每一步都依賴于宿主細胞蛋白:病毒首先結(jié)合細胞表面的硫酸類肝素和葡萄糖胺聚糖;然后囊膜蛋白與CD163結(jié)合使病毒發(fā)生細胞內(nèi)化;最后在網(wǎng)格蛋白介導下形成內(nèi)吞體進入細胞質(zhì)[24-26]。進入細胞質(zhì)的病毒粒子需進一步脫殼釋放出RNA,進行病毒的復制、組裝以及釋放。所以,通過調(diào)控宿主靶向蛋白來抑制病毒復制的思路成為一個新的研究方向[27]。
****.P<0.000 1
圖7 cathepsin L基因敲減抑制PRRSV-GFP復制Fig.7 cathepsin L knockdown inhibits PRRSV-GFP replication
本研究所探究的組織蛋白酶作為細胞中重要的蛋白酶類,同胞外蛋白酶一樣,可以重塑胞外基質(zhì)、降解胞內(nèi)不必要的細胞器及蛋白質(zhì)、降解胞內(nèi)基質(zhì)、傳遞細胞信號、調(diào)節(jié)細胞凋亡[28-32],極有可能在病毒與宿主的相互作用起關鍵作用。因此,未來工作的重點將集中在回答以下問題:1)PRRSV的表面蛋白是否需要經(jīng)細胞cathepsin切割,暴露出真正的抗原表位與細胞受體結(jié)合?2)PRRSV的核衣殼需要哪種cathepsin發(fā)揮功能,才能使位于其中的病毒RNA釋放到細胞核周區(qū)域?3)病毒RNA指導合成的病毒蛋白質(zhì),是否需要細胞cathepsin的功能形成正確的空間結(jié)構(gòu)以有利于子代病毒的組裝?4)具體的分子機制又是如何?
綜上所述,本研究所構(gòu)建的猴源cathepsin基因穩(wěn)定且高效敲減細胞系,為進一步篩選對PRRSV復制有影響的cathepsin奠定了基礎,為研究宿主細胞與PRRSV相互作用的分子機制提供了新的見解,這可能有助于我們進一步了解PRRSV的發(fā)病機制。
本研究利用了RNAi原理,成功構(gòu)建了猴源cathepsin基因的shRNA文庫,并以對PRRSV易感的非洲綠猴胚胎腎上皮細胞Marc145為模型,篩選出了穩(wěn)定敲減細胞株。所構(gòu)建的shRNA文庫中共有45個單克隆,涉及15種cathepsin基因;篩選出的細胞株經(jīng)Q-PCR鑒定,cathepsin基因mRNA的表達量均顯著下降,可以用于探究組織蛋白酶在PRRSV吸附、入侵、脫殼、生物合成、組裝以及釋放中的作用。
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