Fan YANG, Kaiqi LIAN, Lingling ZHOU, Mingliang ZHANG, Yuwei SONG

Tip O/BY/CHA/2010 Şap Hastalığı Virusuna Karşı Artırılmış Duyarlılıkta Süt Emen Fare αυβ8 Transgenik CHO-677 Hücre Hattının Oluşturulması ve Değerlendirilmesi

İntegrin αυβ8 Şap hastalığının ilk aşamasında önemli bir rol oynayabilir ve konak spesifikliği ile doku tropizmini etkileyebilir. Şap hastalığında integrin αυβ8’ınrolünü araştırmak amacıyla stabil olarak süt emen fare αvβ8 heterodimer eksprese eden bir hücre hattı geliştirildi ve CHO-677mαυβ8 olarak adlandırıldı. Yirminci pasajda hücre hattında αv ve β8 integrin subünitlerinin varlığını belirlemek amacıyla sırasıyla polimeraz zincir reaksiyonu ve immunofloresan teknikleri kullanıldı. Sonuçlar bu genlerin hücre hattına başarıyla aktarıldığını ve eksprese edildiğini gösterdi. Şap hastalığı virusunun biyolojik karakterleri hücre hattında belirlendi. Hücre hattı Şap hastalığı virusu tip O/BY/CHA/2010 ile enfekte edildikten sonra hücre hattında viral RNA ve proteinleri parental hücreler ile karşılaştırıldığında belirgin olarak artmıştı. Elde edilen veriler stabil bir hücre hattının başarıyla oluştuğunu gösterdi. Ayrıca, antikor blokajı yönteminin sonuçları, anti-β8 serumu uygulanan hücrelerin plesibo uygulanan hücrelerle karşılaştırıldığında daha düşük virus titresine sahip olduğunu ve böylece Şap hastalığı virusu ile hastalık oluşturmak için gerekli reseptör fonksiyonunda αυβ8’nin oluşturulmasında β8’in önemli olduğunu gösterdi. Bu hücre hattı Şap hastalığı virusunun izolasyonunda, enfeksiyonda integrin αυβ8’in fonksiyonunun ve virusun hücreye girişinin çalışılmasında kullanılabilir.

Establishment and Evaluation of a Suckling Mouse Integrin αυβ8 Transgenic CHO-677 Cell Line with Increased Susceptibility to Type O/BY/CHA/2010 Foot-and-Mouth Disease Virus

Integrin αυβ8 may play an important role in the initial stage of FMDV infection and influence the host specificity and tissue tropism. To study the role of integrin αυβ8 in FMDV infection, a cell line, which stably expressed the suckling mouse αvβ8 heterodimer, was established and designated as CHO-677-mαυβ8. Polymerase chain reaction (PCR) and indirect immunofluorescent assay (IFA) were used to determine the presence of integrin subunits αv and β8 in the cell line at the 20th passage, respectively. The results showed that these genes were successfully transduced and expressed in the cell line. Biological characteristics of FMDV were determined in the cell line. After the cell line was infected with FMDV type O/BY/CHA/2010, there was an obvious increase for viral RNA and proteins, compared with those in the parental cells. Our data showed that a stable cell line was successfully established. Additionally, the results of antibody blockade assay show that the cells treated with anti-β8 serum had a lower virus titre, compared to the mock-treated cells, implying that the β8 was important for maintaining αυβ8 in receptor function required for productive infection by FMDV. The cell line may be used to isolate the FMDV, study the function of integrin αυβ8 in infection and entry of FMDV.

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