論文名稱:

影響單純疤疹病毒第二型之醣蛋白C於感染細胞表面表現補體C3b接受器之因素的探討

 

Analysis of the Factors that Affect the Expression of Glycoprotein C of the Herpes Simplex Virus Type 2 as a C3b Receptor on the Infected Cell Surface

研究生:

陳琬  Woan-Ling Chen

 

(以作者名查詢陽明大學館藏系統)

 

(以作者名查詢全國圖書書目資訊網)

指導教授:

洪善鈴  Shan-Ling Hung

        學位類別:

碩士

        學校名稱:

國立陽明大學

系所名稱:

口腔生物研究所

          學年度:

85

          語文別:

中文

          出版年:

86

關鍵字:

免疫沈  hepes simplex virus type l

 

蛋白  hepes simplex virus type 2

摘要:

單純疤疹病毒第一型(hepes simplex virus type l; HSV-1) 主要引起口腔感染, 而第二型病毒(herpes simplex virus type 2; HSV-2)則主要引起生殖道方面的疾

 

病。這兩型病毒的醣蛋白C(gC-1gC-2) 不論是純化的,或是表現在轉染細胞上的,都具有與補體元素C3b 結合的能力。其實,gC-1gC-2這兩者在與C3b 的結合區域上,具有相似的結構,但是HSV-l 在感染細胞表面上能表現C3b 的接受器(C3b receptor),而HSV-2 雖於感染細胞上表現gC-2,卻無法在感染細胞表面與C3b 結合。然而造成此種差異現象的原困尚不清楚。但在先前的研究結果中發現,有一重組病毒R12-5 ,與野生型病毒HSV-2(HG52) 有不同之表現型,此重組病毒在感染細胞上能表現gC-2,同時也具有和補體元素C3b 結合的能力。因此在本論文中,希望能夠找出導致重組病毒R12-5 與野生型病毒HG52gC-2表現相異的原因。首先,利用免疫沈的方式找出可能與gC-2結合的蛋白質,並且比較從R12-5 HG52兩者的感染細胞萃取物所得到之免疫沈的結果,藉此推測可能影響gC-2於感染細胞表面上表現與C3b 結合能力的蛋白質因子。此外,利用西方墨點法發現HG52R12-5 gC-2大小不盡相同,而進一步利用N-glycosidase 處理作分析。結果發現R12-5 HG52N-基醣化程度上稍有不同。由於R12-5 gC-2本身基因序列的改變,也可能影響其在感染細胞上與C3b 的結合作用,因此藉由序列分析的技術來比較R12-5 與野生型第二型病毒HG52gC-2基因之DNA 序列,結果發現R12-5 DNA 本身發生突變,造成第123 胺基酸serine,第232 胺基酸methionine分別轉變成phenylahninevaline。由以上實驗結果顯示,R12-5 表現型的改變,可能是因為gC-2本身基因的改變,使得突變的gC-2在感染細胞表面上,具有和C3b 結合的能力; 而此種突變,也有可能造成gC-2醣化作用的改變。另外,從免疫沈的結果,可以推測與gC-2結合的蛋白質,可能成為影響感染細胞上的gC-2C3b 結合能力的因子。因此,影響R12-5 gC-2在感染細胞上與C3b 結合能力的因素,有可能是單一的gC-2本身的突變,gC-2的醣化程度,或者是其他蛋白質的影響; 亦有可能是這幾種因素共同造成的結果。

   
 

 Herpes simplex virus type 1 ( HSV-1) mainly causes oral infection; and HSV- 2 causes genital infection. Glycoprotein C (gC) of both HSV-1 and HSV-2 may  modulate the immune system by binding the human complement component C3b. Both  gC-1 and gC-2, as purified proteins or expressed on transfected cells, can  bind C3b. The C3b binding regions of gC-1 are similar to those of gC-2.  However, HSV-I but not HSV-2 expresses a C3b receptor on the infected cell  surface. The reasons and importance of this difference between HSV-1 and HSV-2  infected cells are not clear. According to the previous study, an intertypic  virus, R12-5, which expressed gC-2 on the infected cells had the ability to  bind to C3b on the infected cell surface. This phenotype is different from its  parental HSV-2 strain, HG52. In this thesis, the effects that lead to the  different expressions of gC-2 of R12-5 and HG52. First, the technique of  immunocoprecipitation was used to determine the proteins that might interact  with gC-2. The results of the mmunocoprecipitation from cells infected with  HG52 or other intertypic viruses R12-5 indicated that different proteins are  associated with gC-2 of HG52 and R12-5. These proteins might affect the  binding of gC-2 to C3b on the infected cell surface. In addition, the  molecular weight of gC-2 of HG52 and R12-5 are vaned as determined by Western  blotting. Further analysis using N-glycosidase treatment, N-linked  glycosylation of gC-2 of R12-5 and HG52 are not necessary the same. Because  the mutation on the gC-2 gene of R 12-5 might affect the C3b binding ability,  the DNA sequences of gC-2 gene between HG52 and R12-5 were compared. The  results showed that gC-2 gene of R12-5 had mutations on the amino acids  ofserine (123) and methionine (232) and were changed to phenylalanine and  valine, respectively. These results suggested that the mutated gC-2 on the  infected cells might have the ability to bind to C3b. These mutations may also lead to the change of N-linked glycosylation of gC-2. According to the result    of immunocoprecipitation, one may suspect that different proteins which bound  to gC-2 of HG52 or R12-5 might be the factor that influence the ability of gC- 2 binding to C3b on the infected cell surface. Therefore, the factors that  affect the binding of gC-2 of R12-5 to C3b on the infected cell surface might  be one or multiple, such as mutation of gC-2, changes in glycosylation protein  of gC-2 or other proteins that associated differently with gC-2.