- 學位論文
應用乳酸桿菌表達人干擾素α2b及評估其抗病毒效應
Expression of Human Interferon-α2b by Probiotic Lactobacillus reuteri Pg4 and Evaluation of its Antiviral Activity
摘要
干擾素α為多數真核生物具有的細胞激素，具有抑制多種病毒增殖之能力，該激素在動物細胞受病毒感染後數小時內即產生，並誘導鄰近細胞之抗病毒基因表達，達到抑制病毒之能力。本試驗之目的是將益生菌Lactobacillus reuteri進行基因轉殖，使其帶有人干擾素α2b ( human interferon-α2b, huIFN )基因，之後測定mRNA及蛋白質層次之表現，並評估轉形株對細胞之抗病毒活性。 試驗將huIFN基因構築於乳酸菌-大腸桿菌穿梭載體pNZ3004。以電穿孔 (electroporation) 將重組質體轉殖至腸道益生菌L. reuteri Pg4中，透過載體上的Lactobacillus lactis lacA啟動子和訊號胜肽來調控huIFN表達並且外泌至菌體外。取得轉形菌落後以聚合酶鏈鎖反應（polymerase chain reaction, colony-PCR）確認其帶有huIFN基因並可轉錄huIFN之mRNA。進一步以專一性抗體進行酵素連結免疫吸附分析（enzyme – linked immunosorbent assay, ELISA），將培養16小時之L. reuteri Pg4及其轉形株以ELISA測定huIFN蛋白質表現量。試驗證實，於轉形株之胞內萃取物、細胞壁以及胞外培養液皆可測得huIFN蛋白質，其中以胞內萃取物中表現量為最高。為進一步了解轉形株表現huIFN蛋白質之穩定性，將L. reuteri Pg4及L. reuteri pNZ-huIFN轉形株進行24小時培養，測定生長曲線及huIFN蛋白質表現量，發現L. reuteri Pg4之菌數可於培養後8小時達9 log CFU/mL，然而L. reuteri pNZ-huIFN轉形株需要16小時才能達到將近9 log CFU/mL。在huIFN表現量中發現，於L. reuteri pNZ-huIFN轉形株之胞內萃取物、細胞壁以及胞外培養液中表現量具有相同模式，皆為先升高後下降，且亦以胞內萃取物之表現量最高。在抗病毒試驗中，將純化人干擾素α2b、L. reuteri Pg4及L. reuteri pNZ-huIFN轉形株之胞內萃取物對BHK細胞及293T細胞進行前處理，之後再以Sindbis virus ( SBV ) 進行感染，結果顯示無論在BHK細胞或者293T細胞中，純化人干擾素α2b、L. reuteri Pg4及L. reuteri pNZ-huIFN轉形株皆具有抗SBV活性。 綜觀上述，L. reuteri pNZ-huIFN轉形株可成功表達huIFN，在抗病毒試驗中也證實L. reuteri Pg4及L. reuteri pNZ-huIFN轉形株皆會降低SBV之感染效率，說明此乳酸菌株具有應用於畜禽飼料以做為病毒抑制劑之潛力。
並列摘要
Interferon-α ( IFN-α ) is a cytokine that is able to inhibit various virus, and can be secreted from most eukaryotic cells. After viral infection, IFN-α can be expressed by the infected cells rapidly in few hours, and induces the expression of antiviral genes by cells to block the amplification of virus. The purpose of this study is to genetically modify a probiotic Lactobacillus reuteri strain to express the human IFN-α2b (huIFN), and to evaluate the antiviral activity of the L. reuteri transformants. The DNA sequence of huIFN was amplified by PCR and subcloned into the Lactobacillus expression vector pNZ3004 that was able to secrete recombinant proteins into extracellular medium. After plasmid construction, the recombinant plasmid was used to transform L. reuteri Pg4 by electroporation. Once the L. reuteri transformants were obtained, direct colony PCR was performed to demonstrate the presence of huIFN gene, and reverse transcriptase-PCR analysis was performed to further confirm the transcription of RNA of huIFN. Moreover, the expression levels of the recombinant huIFN expressed by L. reuteri transformants were determined by ELISA during the bacterial growth period. The antiviral activities of the intracellular extract and extracellular medium of the L. reuteri transformants were evaluated in the BHK and 293T cell models of sindbis virus ( SBV ) infection. The results showed that huIFN was successfully expressed by the L. reuteri transformants. More amounts of huIFN were presented in the intracellular extract than in the cell wall pellet or extracellular medium of the L. reuteri transformants. The growth pattern of the L. reuteri transformants was similar to that of the wild type L. reuteri Pg4. The bacterial counts of the L. reuteri Pg4 in the initial fermentation were about 7 log CFU/mL, reached to 9 log CFU/mL after 8 hours of culturing, and remained the stationary phase till 24 hours. The expression levels of recombinant huIFN in the L. reuteri transformants increased with the incubation time, reached the highest levels at 8 hours, and then decreased. In the antiviral experiment, the intracellular extract of the L. reuteri transformants could decrease the replication of SBV in the BHK or 293T cells, similar to the pure huIFN protein. Interestingly, the intracellular extract and extracellular medium of the wild type L. reuteri also showed slight antiviral activity against SBV replication in the BHK or 293T cells. In conclusion, we successfully expressed huIFN by L. reuteri Pg4 and demonstrated the intracellular extract of the L. reuteri transformants did suppress the replication of SBV in cell culture. Therefore, we suggest that the L. reuteri transformants have potential to be used as an antiviral feed additive.
參考文獻
延伸閱讀
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