臺灣博碩士論文加值系統

幽門桿菌利用blood group antigen adhesin (BabA) 附著因子與胃部Lewis b (Leb) 抗原結合，此結合可決定病人胃部菌落量。近來，幽門桿菌SabA (sialyl acid-binding adhesin) 附著因子被發現能與因幽門桿菌感染導致慢性發炎後表現的sLex抗原結合。對於胃部表現微弱或不表現Leb抗原的病人，SabA附著因子可藉由與sLex抗原結合維持幽門桿菌於胃部群居。本研究第一部分在了解幽門桿菌感染MKN45細胞株在發炎狀態下是否能影響sLex抗原表現以及sabA基因是否參與在sLex抗原的表現。MKN45細胞經HP238濾液培養48小時後，以流式細胞儀偵測發現sLex抗原表現量比控制組高出20﹪以上，然而以100 mg幽門桿菌外膜蛋白質濾液做相同實驗並無法增加sLex抗原表現。將帶有不同CT雙核苷酸重複序列表現sabA菌株以同源重組交換方式插入cat基因卡匣構築sabA突變株。利用南方墨點法及西方墨點法驗證三株 (J99, HP307與HP258) 帶有不同CT雙核苷酸重複序列的sabA突變株與一株不帶有CT雙核苷酸重複序列的HP649 sabA突變株。以野生株及突變株製備的濾液培養MKN45細胞48小時後皆能增加sLex抗原表現，但不具有統計差異。
本研究第二部分在於評估SabA附著因子在幽門桿菌貼附MKN45細胞過程中是否扮演重要的角色。四株野生株 (J99, HP307, HP258與HP649) 培養於不同pH值培養液中4小時後，以西方墨點法分析SabA蛋白質表現量。結果顯示SabA蛋白質表現量沒有因不同pH值而受到影響。若將4株野生株及其sabA突變株 (J99, HP307, HP258與HP649) 以同源重組交換方式插入km基因卡匣構築babA突變株及babA-sabA雙基因突變株。只有HP258野生株及sabA突變株成功轉型為babA突變株與babA-sabA雙基因突變株，並以南方墨點法與西方墨點法驗證。經HP238濾液培養48小時的MKN45細胞以野生株及突變株感染1小時後由流式細胞儀檢視細菌貼附細胞情況。SabA附著因子的有無並沒有影響幽門桿菌貼附細胞的能力。將不帶有signal序列的sabA基因 (1.9 kb) 與能區分sabA基因與sabB基因的小片段sabA基因序列 (247 bps) 選殖至pET-30b載體並轉型至大腸桿菌株BL21。誘導表現SabA重組蛋白質 (r-SabA) 並以親合性鎳離子螯合樹脂色層分析法純化後，接種至兔子製備SabA蛋白質專一性抗體。將四株野生株 (J99, HP307, HP258與HP649) 以不同稀釋倍數的抗r-SabA抗體混合反應後感染經HP238濾液培養48小時後的MKN45細胞。以流式細胞儀檢視細菌貼附MKN45細胞，結果顯示不論幽門桿菌有無與抗r-SabA抗體作用並沒有影響幽門桿菌的貼附能力。
基於以上研究，我們發現幽門桿菌所分泌的分子能使MKN45細胞進入發炎狀態並增加sLex抗原表現，而sabA基因與外膜蛋白質並沒有參與在sLex抗原的表現。此外，有無SabA附著因子以及抗r-SabA抗體對於幽門桿菌貼附MKN45細胞並沒有造成影響。

Helicobacter pylori exploits blood group antigen adhesin (BabA) to interact with human gastric Lewis b (Leb) antigen and determines the colonization density in stomach of patients. Recently, SabA adhesin (sialyl acid-binding adhesin) of H. pylori has found to interact with the sialylated Lewix x antigen (sLex), which is expressed after chronic inflammation induced by H. pylori infection. For patients with weak or non-Leb expression, SabA adhesin may interact with sLex to maintain H. pylori colonization on gastric surface. The aim of first part in this study was to understand the relationship among H. pylori infection and expression of sLex under inflammation of MKN45 cells, and determine whether sabA gene involved in the expression of sLex antigen. Fresh MKN45 cells were treated with the filtrate of HP238 for 48 h, and the expression of sLex antigen screened by FACS Calibur was 20% higher than control. However, the filtrate of 100 mg H. pylori outer membrane protein (OMP) extract didn’t stimulate the expression of sLex antigen. Inserting the different CT repeats of wild-type sabA allele with cat cassette were to create different CT repeats of sabA mutants by homologous recombination. Three mutant strains with different CT repeats (J99, HP307 and HP258) and one without CT repeat, HP649 were confirmed by Southern blot analysis and Western blot analysis. Both filtrates of wild-type and sabA mutants treated MKN45 cells for 48 h stimulated the expression of sLex antigen without significant difference.
The aim of second part was to evaluate the role of SabA adhesin in H. pylori adhesion to MKN45 cells. Four wild-type strains (J99, HP307, HP258 and HP649) were incubated in different pH conditions for 4 h and then the expression of SabA protein was analyzed by Western blotting. The expression of SabA protein was not affected in different pH conditions. Inserting the babA alleles of 4 wild-type and mutants (J99, Hp307, HP258 and HP649) with km cassette to create babA mutants and babA-sabA double mutants by homologous recombination. Only wild-type and sabA mutant of strain HP258 were transformed to babA mutant and babA-sabA double mutant successfully and confirmed by Southern blot analysis and Western blot analysis. MKN45 cells infected with wild-type strains and mutants for 1 h after cell pretreated with the filtrate of RPMI 1640 or HP238 for 48 h and then the adhesion of bacteria was screened by FACS Calibur. No difference was found between wild-type strain and mutant H. pylori adhesion to MKN45 cells. The sabA gene (1.9 kb) without the signal sequence and a small fragment (247 bps) of sabA gene, which can distinguish sabA from sabB, were constructed into pET-30b vector and transformed to E. coli BL21. The recombinant SabA (r-SabA) protein was expressed and purified by Ni2+ column chromatography. The r-SabA proteins were injected to rabbit to develop the SabA antibody. Wild-type bacteria (J99, HP307, HP258 and HP649) pretreated with different dilution of anti-r-SabA antibody and then infected MKN45 cells after cell pretreated with filtrate of HP238 for 48 h. The result indicated anti-rSabA antibody had no effect in H. pylori adhesion.
Base on the above results, we find that H. pylori component can stimulate the expression of sLex antigen under inflammation of MKN45 cells whereas sabA gene and OMP of H. pylori do not involve in the expression of sLex antigen. In addition, SabA doesn’t affect H. pylori adhere to MKN45 cells, and anti-r-SabA antibody has no effect in H. pylori adhesion.

中文摘要 i
英文摘要 iii
致謝 v
目錄 vi
表目錄 viii
圖目錄 ix
符號與縮寫 xi
緒論 1
材料與方法 20
一、菌種來源、保存與培養 20
二、細胞株的培養與保存 20
三、聚合酶連鎖反應 (PCR) 21
四、洋菜膠體電泳 21
五、核酸定序分析 21
六、細菌DNA的抽取 22
七、sabA變異株之構築 23
八、南方墨漬雜交法 (Southern blotting hybridization) 25
九、SDS-PAGE蛋白質膠體電泳 26
十、西方墨點法 (Western blotting) 27
十一、抽取幽門桿菌外膜蛋白質 (OMP) 27
十二、MKN45細胞株之Sialylation醣化作用 27
十三、量測不同pH值下幽門桿菌SabA蛋白質表現程度 28
十四、貼附能力試驗 (Adhesion assay) 29
十五、重組蛋白質之誘導表現 30
十六、親合性鎳離子螯合樹脂色層分析法純化重組蛋白質 30
十七、製備多株抗體 (Polyclonal antibody) 32
十八、利用ELISA方式測定兔子血清抗重組蛋白質效價 32
十九、抗rSabA多株抗體影響幽門桿菌貼附能力試驗 33
二十、細胞表面抗原及細菌貼附數目分析 33
結果 34
第一部份：幽門桿菌感染與唾液酸化路易斯抗原x表現之相關性 34
一、幽門桿菌對於細胞發炎狀態中引發sLex抗原表現的影響 34
二、構築sabA基因突變株 36
三、探討sabA基因對於細胞sLex抗原表現量的影響 38
第二部份：探討幽門桿菌附著因子SabA蛋白質貼附細胞能力及影響程度 39
ㄧ、幽門桿菌SabA蛋白質在不同pH值的表現量 39
二、構築babA基因突變株與babA-sabA雙基因突變株 40
三、探討SabA附著因子對於幽門桿菌貼附細胞能力的影響 41
四、大量表現SabA蛋白質與製備抗rSabA多株抗體 42
五、探討抗rSabA多株抗體對於幽門桿菌貼附作用 (Adhesion) 的影響 44
討論 46
參考文獻 52
圖表 67
附錄 90
自述 108

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