TW202124717A - 人4IgB7-H3的突變編碼基因及其調節免疫的應用 - Google Patents
人4IgB7-H3的突變編碼基因及其調節免疫的應用 Download PDFInfo
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Abstract
本發明公開一種新的人4IgB7-H3的突變編碼基因及其調節免疫的應用,其核苷酸序列如SEQ ID NO: 1所示。該人4IgB7-H3的突變編碼基因比野生型基因的生物學功能增强,即突變編碼4IgB7-H3與抑制性受體結合對T細胞的增殖、活化的抑制作用增强。對4IgB7-H3作爲共刺激分子在T、B細胞活化、輔助T細胞分化,信號轉導及作用細胞的細胞激素分泌過程中產生重要作用。對腫瘤逃避免疫監視中難以誘導腫瘤特異性T細胞活化的主要原因產生了解決及治療作用。其在製備基因組工程、基因重組、以T細胞和樹突狀細胞爲基礎的治療惡性腫瘤和傳染性疾病的免疫治療藥物、疫苗以及蛋白替補療法、緩解過敏等中有突出而廣泛的應用前景。
Description
本發明關於生物醫藥免疫療法領域,尤其涉及一種人4IgB7-H3的突變編碼基因及其調節免疫的應用。
正性/負性協同刺激信號(又稱共刺激信號,Co-stimulatory signals)之間的有序調節達到的動態平衡在機體抵抗外來抗原的入侵以及防止自體免疫性疾病的發生中產生重要的作用。T淋巴細胞(簡稱T細胞)的活化或增殖依賴於正性/負性雙重信號,其對T細胞抗原特異性的有效激發以及免疫效應適時中止是必須的。免疫調節失去平衡、不管抑制或者過度活化,都屬免疫異常,從而影響機體的免疫反應,引起的疾病即爲各種免疫性疾病,包括腫瘤、發炎、自體免疫性疾病等。另一方面,T細胞活化與增殖依賴於雙信號:一是抗原呈現細胞(APC)呈現的抗原肽-人類白血球抗原(HLA)複合物,二是共刺激信號(第2信號)。APC呈現的抗原肽-人類白血球抗原(HLA)複合物由特殊T細胞抗原受體(TCR)識別,並將信號傳遞給T細胞,該信號對T細胞活化是必需的,但不引起T細胞增生和分泌細胞激素。共刺激信號爲T細胞抗原特異性活化所必需,啓動、維持並調節活化級聯反應決定了T細胞是活化增殖、抑或轉變爲無反應狀態(anergy)甚至凋亡(apoptosis)。雙信號學說(two-signal model)的提出,解釋了成熟外周B細胞如何識別抗原,以及T細胞如何獲得來自於APC呈現的抗原肽-主要組織相容性複合物(major histocompatibility complex I,MHC)信號,因此共刺激信號已經成爲人們研究的熱點。共刺激信號爲抗原(Ag)非特異性,除影響T細胞的活化與增殖外,還直接影響細胞激素的産生。共刺激分子在腫瘤免疫中產生重要作用。研究發現大多數腫瘤細胞表現MHC-1類分子和腫瘤Ag,是潛在的APC,但免疫原性較弱,因爲其不表現或低表現B7-1,故難以誘導腫瘤特異性T細胞活化,這是腫瘤逃避免疫監視的重要原因之一。因此,可以透過增加或增强共刺激信號,達到治療腫瘤的作用。
由此可見,腫瘤免疫逃逸是腫瘤發生、侵襲及轉移的重要機制。由於腫瘤細胞通常不能提供有效的抗原信號,自身具備抵抗免疫作用細胞攻擊的能力,或者機體存在免疫反應缺陷及免疫抑制,這些情况均導致了腫瘤細胞逃避機體的免疫監控。在這一過程中,協同刺激分子(又稱共刺激分子)及其調節網路發揮著重要作用。協同刺激分子是T淋巴細胞活化過程中一類重要的調節分子,共刺激分子與其受體或配體作爲共刺激信號,在T、B細胞活化、輔助T細胞分化、信號轉導及作用細胞的細胞激素分泌過程中產生重要作用。協同刺激分子主要分爲B7/CD28和TNF/TNFR兩個超家族。經典的B7/CD28信號途徑主要以協同刺激的形式來調節初始T細胞的活化。然而,新近發現的一些B7家族新成員亦可透過作用於活化的T細胞,對免疫反應產生負性調控作用。B7家族屬免疫球蛋白類,爲50000~70000d的跨膜糖蛋白,B7免疫蛋白家族的成員包括B7 - 1、B7 - 2、B7 - H1(即PD - L1)、B7 - H2、B7 - H3和PD - L2。它們結構相似均可在機體免疫過程中作爲共刺激信號,參與T、B細胞的活化和機體免疫。因此,B7家族在腫瘤治療、器官移植和自體免疫疾病的治療方面有重要作用,在基礎研究方面則可以用於T、B細胞分化、活化機制、抗原呈現、共刺激機制、免疫耐受、移植排斥反應和自體免疫等的研究。
B7-H1、B7-H3就是其中兩個重要的負性調控協同刺激分子。陳陸俊等首次報導了負性協同刺激分子B7-H1、B7-H3在結直腸癌中的表現及其臨床意義(陳陸俊,蘇州大學碩士學位論文,2009,協同刺激分子B7-H1和B7-H3在結直腸癌中表現的臨床意義及調控機制),證實它們在腸癌的發生發展中發揮重要作用並負性調控T淋巴細胞在腫瘤組織中的浸潤,檢測B7-H1、B7-H3分子可作爲腸癌診斷和預後判斷的重要生物學指標。同時,腫瘤微環境中的細胞激素IFN-γ和TNF-α對腫瘤細胞表現協同刺激分子B7-H1、B7-H3產生重要的調控作用。B7-H3分子可透過膜型和可溶性(又稱SB7-H3)兩種形式在腸癌的腫瘤免疫逃逸過程中發揮作用。
人B7-H3,也稱爲CD276,是屬B7 - CD28途徑的免疫檢查點分子。B7 - H3是一種I型跨膜蛋白,在小鼠中由9號染色體編碼,在人類中由15號染色體編碼。B7-H3作爲B7家族的最新成員,mRNA廣泛存在於非淋巴組織,在許多淋巴器官如脾、淋巴結、胸腺、嬰兒的肝和胸腺也有表現。此外,部分腫瘤細胞株如黑色素瘤G361、子宮頸腺瘤、HeLa S3、慢性骨髓性淋巴瘤K562、肺癌A546和結直腸腺癌SW480中,B7-H3的mRNA也有表現。其受體在活化的T細胞表面可被誘導表現,B7-H3對CD4+和CD8+細胞的生成均具有增加作用,並能誘導CTL産生,並可選擇性地增加IFN-γ的生成,參與T、B細胞的活化和機體免疫,在腫瘤治療、器官移植和自體免疫疾病的治療方面具有重要的作用(Suh WK, et al. The B7 family member B7-H3 Prefeerntialy down-regulates T helper type1-mediated immune responses[J]. Nat Immunol, 2003, 4(9): 899-906.)。B7-H3不能與CTLA-4 ICOS和PD-1結合,表明B7-H3有受體或配體,但目前還沒有分離到。
在人類中,B7 - H3基因具有四個Ig樣重複,這些重複可以交替剪接以産生包含四個Ig樣結構域(4Ig)或兩個Ig樣結構域(2Ig)的蛋白質,即異構體4IgB7-H3(或B7-H3b)和2IgB7-H3。4IgB7-H3帶有兩個拷貝的IgV- IgC結構域,爲人大部分細胞和組織中的主要表現形式,其胞外段兩對VC之間鹼基重複率爲98%,是2IgB7-H3基因複製的結果。孫靜等(孫靜,蘇州大學博士學位論文,2010,協同刺激分子B7-H3兩種異構體的基因演化及其生物學功能差異的研究)根據B7-H3分子2種異構體在不同物種中的分布及其基因複製模式的研究、人2IgB7-H3和4IgB7-H3蛋白質的空間結構模擬及其受體表現分析、及人2IgB7-H3和4IgB7-H3生物學功能差異研究等發現:1、4IgB7-H3是2IgB7-H3基因複製的結果,除表現於人類外還可表現於豚鼠、家犬、非洲象、牛、熊猫、蝙蝠及高級靈長類動物如猩猩、猴體內;2、人2IgB7-H3和4IgB7-H3兩種蛋白質具備不同的空間構象;3、活化的T細胞上可表現兩種受體與B7-H3的2種異構體相結合;人2IgB7-H3與小鼠B7-H3與刺激性受體相結合,發揮促進T細胞的活化與增强單核細胞的功能;而人4IgB7-H3可與抑制性受體結合,對T細胞的活化具有抑制作用。
在人4IgB7-H3分子「PQRSPT」模體的生物學特性的研究發現在外顯子複製過程中産生的保守性氨基酸「PQRSPT」可成爲人4IgB7-H3不能形成可溶性形式及對活化T細胞産生抑制作用的重要模體。蛋白質建模發現氨基酸高度重複的2種異構體具備不同的空間構象,融合蛋白結合實驗推測其可結合不同的受體發揮生物學功能。體外實驗對活化T細胞和單核細胞的作用證實了之前的實驗結果。人2IgB7-H3與小鼠B7-H3基因可促進活化T細胞的增殖,增强單核細胞的作用;人4IgB7-H3主要表現於抗原呈現細胞中可發揮抑制T細胞活化的生物學功能。
綜上,人B7-H3在腫瘤治療、器官移植和自體免疫疾病的治療方面具有重要的作用,且其異構體2IgB7-H3與4IgB7-H3在功能上相互配合,增加了該信號通路的重要性和複雜性,且由於B7-H3受體或者配體尚未被發現,因此關於其功能學研究的報導還存在很大的爭議。圍繞B7-H3兩個異構體開展深入研究對揭示共刺激分子在免疫反應中的精確調控具有重要的生物學意義。而提供2IgB7-H3與4IgB7-H3的各種突變體及功能研究,也爲上述B7-H3的功能研究提供新途徑,但目前尚未有4IgB7-H3的突變體或4IgB7-H3的突變編碼基因的報導。
本發明提供了一種新的人4IgB7-H3的突變編碼基因,比野生型4IgB7-H3基因具有增强的生物功能,爲抑制過度或異常活化的免疫,特別是T淋巴細胞介導的細胞免疫及T淋巴細胞的發育、或與功能增强的2IgB7-H3突變體的配合提供一種新的技術手段;尤其是透過新的人4IgB7-H3的突變編碼基因、其編碼的蛋白或其受體或配體、相關的例如RNA分子等藥物或含其的藥物組合物來干預免疫逃逸,從而調節或改善受試者的免疫反應;同時提供在調節免疫的應用,例如其在製備預防或治療惡性腫瘤和傳染性疾病的免疫治療藥物、疫苗以及緩解過敏藥物等中的應用。
爲解決上述技術問題,本發明的技術手段之一爲:提供一種人4IgB7-H3的突變編碼基因,其中,所述人4IgB7-H3的突變編碼基因的核苷酸序列如SEQ ID NO: 1所示。
眾所周知,DNA是絕大多數生物的遺傳物質,DNA攜帶的遺傳資訊、DNA複製、DNA修復等過程對維持染色質穩定和基因表現、調控具有重要作用;按照中心法則DNA轉錄爲mRNA,並轉譯爲蛋白質,執行相應的生理功能。一方面,細胞內mRNA的轉錄在時間和空間上受到了多層次的精細調控;另一方面,最新實驗研究發現了細胞內另一反饋調節機制,即RNA透過調控Mediator的相分離對轉錄過程進行反饋調節。基因(DNA分子)最主要的功能是編碼功能性蛋白質産物或RNA産物。其中,蛋白質産物可以是大分子的結構蛋白、蛋白酶、蛋白質聚合體、核孔複合物(NPC)等,也可以是氨基酸、多肽(多肽鏈)等;RNA産物則可以是核酶和tRNA、rRNA、mRNA等。雖然之前技術顯示蛋白質折疊等蛋白質二級以上結構主要由一級結構中的氨基酸序列決定,但最新的研究例如大腸桿菌中的氯黴素乙醯基轉移酶的同義突變研究則顯示基因層面對於蛋白質二級以上結構可能也有影響;另一方面,一級結構氨基酸序列相同的蛋白質構象也有可能不一樣,類似於小分子化合物有手性,大分子也會有不同的空間構象,且可能具有不同的活性。而本發明涉及基因層面,故可能對於中心法則中的上述RNA、蛋白質等物質及物質結構,基因表現、轉錄等屬性,以及基因藥物等應用方面均能産生影響。
根據本發明,所述的突變編碼基因屬外顯子序列,較佳地用於轉錄mRNA,於5ʼ端第2142位點進行了雜合突變C>T。突變通常分爲同義突變和錯義突變。經典理論認爲,錯義突變導致蛋白質分子中某一氨基酸爲另一氨基酸所替代;而同義突變(使用同義密碼子)改變了基因編碼序列,卻保留了編碼的氨基酸序列,由此不會影響到其所轉譯的蛋白質的正確折疊過程以及蛋白質的結構和功能,因此被稱爲「沉默突變」。然而,近期的研究證實,罕見的同義密碼子往往比普通密碼子轉譯得慢,其改變了核糖體合成蛋白質的速度,由此不僅會擾亂蛋白質折疊,導致更容易降解的結構形成,造成細胞生長缺陷。越來越多的研究表明,同義突變可以透過多種機制改變蛋白質量、轉譯準確性、轉譯後修飾、分泌效率和蛋白質的最終折疊結構,從而顯著改變體內蛋白質進而影響細胞的功能。因此,同義突變的研究對有效的蛋白質設計和與同義突變相關疾病的治療將産生廣泛的影響。
如上所述,本發明的突變編碼基因可能對於4IgB7-H3的二級、三級蛋白質構象産生影響,而目前4IgB7-H3在免疫調節中作爲受體還是配體作用尚未有定論。故爲解決上述技術問題,本發明的技術手段之二爲:提供如上所述的突變基因編碼的蛋白或其受體或配體;其中,當本發明突變基因編碼的蛋白爲受體時,與其特異性結合的爲配體;而當本發明突變基因編碼的蛋白爲配體時,與其特異性結合的爲受體。較佳地,所述蛋白的第一個C樣結構域末端包含PQRSPT模體,該模體可能是人4IgB7-H3不能形成可溶性形式的一個重要模體。所述蛋白可爲膜型蛋白。爲解決上述技術問題,本發明的技術手段之三爲:提供一種重組表現載體,其中,所述重組表現載體包含如上所述的人4IgB7-H3突變編碼基因。
較佳地,所述重組表現載體爲病毒表現載體或原核表現載體或真核表現載體或反轉錄病毒載體或腺病毒載體或單純孢疹病毒。
更佳地,所述病毒表現載體爲慢病毒表現載體或腺相關病毒表現載體。
爲解決上述技術問題,本發明的技術手段之四爲:提供一種宿主細胞,其中,所述宿主細胞包括如上所述的重組表現載體。
較佳地,所述宿主細胞爲真核細胞,更佳為哺乳動物細胞。
爲解決上述技術問題,本發明的技術手段之五爲:提供一種分離的mRNA,其中,所述mRNA由如SEQ ID NO: 1所示的核苷酸序列轉錄而得。較佳地,所述mRNA爲IVT mRNA。
本發明所述的體外轉錄信使RNA (in vitro
transcription messenger RNA, IVT mRNA) 是在體外條件下以DNA爲模板轉錄得到的一種mRNA,這種mRNA可以指導特定蛋白質的合成,阻止或改變某種特定的疾病。因此,IVT mRNA可以作爲一種傳遞遺傳資訊的潛在新藥。這種IVT mRNA能夠更好地控制轉錄基因、轉錄過程與蛋白功能等。IVT mRNA可以將特定的遺傳資訊導入患者的細胞內,快速表現出蛋白質,從而阻止或改變某種疾病。目前,IVT mRNA免疫療法主要應用於三個領域:惡性腫瘤的免疫治療、治療傳染性疾病的疫苗、緩解過敏的疫苗。
IVT mRNA有2條遞送途徑。一條是將IVT mRNA導入患者離體細胞,再將轉染的細胞有選擇性地導回患者體內。這種方法適用於基因組工程、基因重組、以T細胞和樹突狀細胞 (dendritic cells,DC) 爲基礎治療惡性腫瘤和傳染性疾病的免疫療法以及一些蛋白替補療法等。另一條途徑是透過多種方式直接遞送IVT mRNA,包括肌肉注射、真皮內注射、結內注射、靜脈注射、皮下注射、氣管內滴入和鞘內注射等。這種方法適用於腫瘤、傳染性疾病的治療、治療過敏的免疫耐受養生法以及其他蛋白替補療法等。對於疫苗而言,IVT mRNA具有的强烈免疫刺激作用和固有的佐劑特性有很多優勢,它能夠引起有效的抗原特異性細胞和體液免疫反應。
細胞內RNA感應器的識別機制和mRNA免疫識別結構元件:
IVT mRNA透過活化模式識別受體 (pattern recognition receptor,PRR) 引起各種下游效應,從而識別病毒RNA,引起免疫反應。
在免疫細胞中,Toll樣受體3 (Toll-like receptor 3,TLR3)、TLR7和TLR8存在於胞內體之中。細胞吞噬IVT mRNA後能夠活化TLR,並且引起干擾素分泌。其中,TLR3用於識別雙股RNA (double-stranded RNA,dsRNA),TLR7和TLR8用於識別單股RNA (single-stranded RNA,ssRNA)。最有效的干擾素誘導劑聚尿苷酸透過TLR7發揮作用。而在非免疫細胞中,大多數干擾素是透過活化細胞質基質受體産生的。另外,細胞質中的RNA感應器能夠影響免疫刺激作用和mRNA的轉譯能力。它可以抑制蛋白質轉譯,降解RNA,直接産生抗病毒活性。IVT mRNA發揮藥效的部位主要是細胞質。與天然mRNA不同,IVT mRNA由細胞外進入細胞質,其轉譯並修飾後産生相應的具有藥理活性的蛋白質。在免疫治療中,IVT mRNA的藥效取決於編碼蛋白的處理加工過程。與內源性蛋白相似,mRNA編碼産生的蛋白分子會被蛋白酶體降解爲肽片段,然後MHC-I類分子將這些肽片段呈現給CD8+ T細胞,誘導T細胞免疫反應。一般來說,胞內蛋白不會與MHC-II類分子結合引起輔助T (helper T,Th) 細胞反應。但如果將分泌信號導入抗原編碼序列,抗原蛋白在分泌信號作用下就可以重新導向細胞外,與MHC-II類分子結合,從而同時誘導Th細胞反應。
由此,爲解決上述技術問題,本發明的技術手段之六爲:提供一種細胞,包含如上所述的分離的mRNA和RNA感應器和/或mRNA免疫識別結構元件;較佳地,所述細胞包括免疫細胞和非免疫細胞;更佳地,所述免疫細胞包括樹突狀細胞和淋巴細胞,所述淋巴細胞包括B細胞和T細胞,所述T細胞包括輔助性T細胞、抑制性T細胞、作用T細胞、細胞毒性T細胞、遲發性變態反應T細胞、天然T細胞和記憶T細胞;其中,輔助性T細胞分爲Th1、Th2、Th17或Treg。
爲解決上述技術問題,本發明的技術手段之七爲:提供一種分離的miRNA,其中,所述分離的miRNA標靶SEQ ID NO: 1和/或SEQ ID NO: 2所示的核苷酸。較佳地,所述miRNA藥物標靶SEQ ID NO: 1和/或SEQ ID NO: 2所示的核苷酸的第2142位點。所述分離的miRNA可以作爲本發明突變編碼基因或其編碼蛋白關聯疾病的檢測標記物,也可以應用於防治相關疾病,例如腫瘤。
爲解決上述技術問題,本發明的技術手段之八爲:提供一種標靶藥物,所述標靶藥物標靶本發明上述人4IgB7-H3的突變編碼基因,所述標靶藥物爲反義RNA或siRNA或環狀RNA(circRNA)或lncRNA或shRNA等不編碼RNA(Non-coding RNA,ncRNA);或者標靶本發明突變編碼基因編碼的蛋白或其受體或配體、所述標靶藥物例如抗體。所述標靶藥物可抑制本發明4IgB7-H3的突變編碼基因、或其編碼蛋白、或該蛋白的受體或配體,干預其生物功能;或者用於檢測上述物質。
其中,所述抗體透過在細胞內的內化和攝取情况來進行生物藥物抗體的選擇和最佳化。
目前的分子生物學研究顯示,在基因表現調控和基因轉錄等過程中,除了DNA,RNA也發揮著重要的作用,RNA剪接、降解、代謝和化學修飾等都與細胞功能息息相關,每種RNA修飾都需要其特異的調控因子,包括修飾酶、去修飾酶和識別蛋白。特別是非編碼RNA(non-coding RNA,ncRNA)在細胞各項生物過程中發揮著不可忽視的作用,被喻爲生命的「暗物質」,其包括但不僅限於rRNA、tRNA、snRNA、IncRNA、snoRNA 、circRNA和miRNA 等多種已知功能的RNA,還包括未知功能的RNA。其中,circRNA與傳統的線性RNA不同,circRNA分子呈封閉環狀結構,不受RNA外切酶影響,表現更穩定,不易降解。在功能上,近年的研究表明,circRNA分子富含miRNA結合位點,在細胞中產生miRNA海綿(miRNA sponge)的作用,進而解除miRNA對其目標基因的抑制作用,升高目標基因的表現程度。透過與疾病關聯的miRNA相互作用,circRNA在疾病中發揮著重要的調控作用。由此,標靶本發明突變編碼基因、其編碼蛋白或其相關miRNA的circRNA也是診斷或防治免疫性疾病的重要手段。
爲解決上述技術問題,本發明的技術手段之九爲:提供一種藥物組合物,其中,其包括如上所述的人4IgB7-H3突變編碼基因、所述的蛋白或其受體或配體、所述的重組載體、所述的宿主細胞、所述的分離的mRNA、所述的細胞、所述的miRNA藥物或標靶藥物。較佳地,所述藥物化合物包括藥學上可接受的載體或賦形劑。
爲解決上述技術問題,本發明的技術手段之十爲:提供一種如上所述的人4IgB7-H3的突變編碼基因、所述的蛋白或其受體或配體、所述的重組載體、所述的宿主細胞、所述的分離的mRNA、所述的細胞、所述的分離的miRNA、所述的標靶藥物或所述的藥物組合物在製備調節T細胞活性和/或調節T細胞增殖的藥物中的用途;進一步地,提供其在製備調節免疫或免疫治療的藥物中的用途;較佳地,所述調節免疫或免疫治療的藥物爲預防和/或治療腫瘤、傳染性疾病或緩解過敏的藥物。
其中,所述調節免疫或免疫治療的藥物爲疫苗。
所述腫瘤爲胃癌、腸癌、肝癌、肺癌、膀胱癌、睪丸癌、前列腺癌、子宮頸癌、乳房癌和淋巴癌中的一種或多種。
根據本發明,所述的「免疫調節」是指對過度或異常活化的免疫反應進行抑制,而對過低或被抑制的正常免疫反應進行促進或活化。
本發明所用試劑和原料均市售可得。
本發明的積極進步效果在於:
轉染了本發明人4IgB7-H3突變編碼基因的HEK293T細胞比轉染了野生型基因的細胞增殖快,說明突變基因比野生型基因具有更强的生物學功能。換言之,該人4IgB7-H3的突變編碼基因比野生型基因的生物學功能增强,即突變編碼4IgB7-H3與抑制性受體或配體結合對T細胞的增殖、活化的抑制作用增强。從而可用於抑制過度或異常活化的免疫、或與2IgB7-H3及其突變體的配合調節免疫,在腫瘤治療、器官移植和自體免疫疾病的治療方面有廣泛的應用前景。此外,該突變編碼基因還可以作爲相關疾病的診斷指標,並由此設計有效的疾病干預手段。突變編碼基因編碼的蛋白或其受體或配體、針對突變編碼基因的mRNA及含其的細胞、miRNA和siRNA、抗體等標靶藥物也可以用於腫瘤治療、傳染病治療、緩解過敏、器官移植和自體免疫疾病的臨床應用。
本申請案請求申請日爲2019/12/30的中國專利申請案201911399571.6,以及申請日爲2020/9/10的中國專利申請案202010949660.X的優先權。本申請案引用上述中國專利申請案的全文。
下面透過實施例的方式進一步說明本發明,但並不因此將本發明限制在所述的實施例範圍之中。下列實施例中未註明具體條件的實驗方法,按照習知方法和條件,或按照商品說明書選擇。
實施例1 含4IgB7-H3野生型編碼基因和4IgB7-H3突變編碼基因的質體的製備
1、材料和方法
將人工合成的野生型4IgB7-H3編碼基因(SEQ ID NO: 2,NCBI Reference Sequence: NM_001024736.1)和4IgB7-H3突變編碼基因(SEQ ID NO: 1)進行PCR後,分別構建到PGMLV-PA7和PGMLV-PA6,並分別以Blasticidin和puro爲篩選標記。載體以本領域習知技術轉化到勝任細胞中,將勝任細胞塗盤,倒置於37℃恆溫培養箱(上海一恆科學儀器有限公司)內過夜培養。第二天挑選單株,每一種載體株挑選兩個送定序公司進行定序鑒定,經過比對,重組株中插入片段序列與目的片段序列完全一致,因此質體構建成功。獲得人4IgB7-H3野生型編碼基因(SEQ ID NO: 2)及人4IgB7-H3(SEQ ID NO: 1)的突變編碼基因。流式細胞實驗驗證目的基因的表現程度。具體的轉化、定序和流式細胞實驗驗證目的基因的表現程度過程如下。
(1)轉化
A. 將勝任細胞置於冰上(4℃)待其自然解凍後,取10 µl的連接産物加入勝任細胞中於冰上(4℃)放置30 min。
B. 之後於42℃水浴中熱擊90 S。然後迅速置於冰上(4℃)放置2-3 min。
C. 加入500 µL的不含抗生素的SOC培養基於37℃、225 rpm下振蕩培養45 min。
D. 以3000轉離心2 min,棄掉900 µL的上清液,將管底的菌液吹打散開,加入到含有載體上對應抗性(氨苄或卡那等)的培養平板中,用滅菌的塗布器塗勻,倒置於37℃恆溫培養箱內過夜培養。
(2)定序
每個株挑選兩個送定序公司進行定序鑒定,經過比對,重組株中插入片段序列與目的片段序列完全一致,因此質體構建成功。獲得含人4IgB7-H3野生型編碼基因(SEQ ID NO: 2)及人4IgB7-H3(SEQ ID NO: 1)的突變編碼基因的質體,分別命名爲CMV-4IgB7-H3(CD276)WT-PGK-puro(11,459bp,質體圖譜見圖2,以下簡稱4IgB7-H3(CD276)WT質體)和CMV-4IgB7-H3(CD276)MT(c.C2142T)-PGK-Blasticidin (11,267bp,質體圖譜見圖1,以下簡稱4IgB7-H3(CD276)MT質體)。
(3)流式細胞實驗驗證目的基因的表現程度
細胞:HEK293T;DMEM+ 10% 胎牛血清(GIBICO);HG transgene reagent(吉滿生物科技);0.25% Trypsin(GIBICO);高純度質體抽提套組(QIAGEN);熒光顯微鏡(Olympus);異丙醇;流式抗體;生物安全櫃(Thermo);CO2
培養箱(Thermo),BD FACSCalibur流式細胞儀。
A. 過表現質體暫態轉染目標細胞
a. 將狀態良好,處於對數生長期的空白血球用0.25%胰酶(GIBICO)消化,用完全培養基懸浮成單細胞懸浮液,細胞計數後,按照每孔5×105
個細胞接種於35 mm培養皿(上海前塵生物)中;
b. 培養過夜,觀察細胞生長狀態。在細胞覆蓋率80%左右時,進行轉染實驗;
c. 轉染2 h前換液爲新鮮的培養基。
d. 轉染
取無菌的1.5 ml EP管,轉染體系按下表:
混勻後,室溫放置15 min-20 min後,均勻滴加到提前換過液的培養皿中,後置於CO2
培養箱中培養。
無血清的DMEM | 200 μl |
過表現質體 | 2 μg |
HG transgene reagent | 6 μl |
B. 透過流式抗體細胞染色檢測目的基因的表現
a. 轉染72 h後,吸除上層培養基,以適當PBS清洗細胞,後加入適當胰酶消化、終止。使用血球計數盤計數,調整細胞密度至1×106
cells/ml,以每管100 μl的細胞液分裝到流式管中;
b. 加入適當體積熒光標記的一抗到已經準備好的細胞懸浮液裡,在4℃下避光孵育20 min;
c. 加入1 ml的PBS清洗細胞,以300×g 離心5 min,小心吸除上清液,重複此步驟1次;
d. 用300 μl的PBS重新懸浮細胞;
e. 流式細胞儀分析。
結果:如圖3所示,經流式驗證,4IgB7-H3(CD276)WT質體和4IgB7-H3(CD276)MT質體與對照組相比,表現量升高,啓動子正常啓動,有過表現效果,由此說明4IgB7-H3 (CD276)WT表現質體和4IgB7-H3 (CD276)MT表現質體構建成功,可以進行後續的病毒包裝。
實施例2 慢病毒的包裝和病毒滴度的測定
細胞株:HEK 293T,慢病毒的包裝細胞,爲貼壁依賴型成上皮樣細胞,生長培養基爲DMEM (含10% FBS)。貼壁細胞經培養生長增殖形成單層細胞。
菌株:大腸桿菌菌株DH5α。用於擴增慢病毒載體和輔助包裝載體質體。
慢病毒包裝系統:將構建成功的慢病毒重組質體和包裝質體採用Qiagen公司的質體抽提套組提取。所得的質體DNA溶於無菌的TE中,以紫外光吸收法測定其濃度及純度,保證所提質體DNA的A260/A280在1.8~2.0之間。
一、慢病毒包裝
1) HEK 293T細胞分盤:轉染前一天,將已經長好的細胞以合適的比例繼代到10 cm的培養皿中,當細胞長到70%~80%時準備轉染。
2) 轉染前換液:轉染前1~2 h將需要轉染的細胞換新鮮的培養基,12 ml/10 cm的皿。
轉染:取無菌的1.5 ml EP管或15 ml離心管,轉染體系按下表:
混勻後,室溫放置15 min-20 min後,均勻滴加到提前換過液的培養皿中,後置於CO2
培養箱中培養。
3) 加Enhancing buffer:轉染10~12 h後,均勻滴加100×Enhancing buffer促進轉染,120 μl/皿。
4) 換液:轉染18~20 h後,小心吸掉細胞培養液棄於盛有消毒液的廢液杯中,然後加15 ml新鮮的細胞培養基繼續培養。
5) 病毒收集:換液48 h後,吸取細胞上清液於50 ml離心管,4℃,4,500×g離心5 min,上清液用0.45 μm過濾器過濾後轉移到新的離心管中,最後將濾液分批轉移到濃縮裝置中,4℃,4,500×g,離心10 min,棄下層的液體於盛有消毒液的廢液杯中,最後一次4℃,4,500×g,離心20 min,此時過濾器上層中的液體即爲病毒濃縮液。
6) 病毒分裝與保存:將病毒分裝後保存於-80℃。
DMEM | 1 ml |
質體 | 10 μg |
Lenti-HG Mix | 10 μl(10 μg) |
HG transgene reagent | 60 μl |
二、Lentivirus滴度測定
樣品製備:將HEK 293T細胞培養至對數生長期,病毒稀釋用培養液爲含10%FBS的細胞培養基。第一天,細胞胰酶消化計數後,按照每孔2×105
細胞接種12孔盤,37℃培養過夜,感染時細胞長至20~40%的融合密度;感染前換液,感染前1~2 h將需要感染的細胞換新鮮的培養基,0.91 ml每孔,Polybrene濃度爲1 µg/ml。第二天,對12孔盤當天轉染時,將保存於-80度冰箱中的病毒液冰浴融化,用含有10% FBS的細胞培養液進行梯度稀釋。選取所需的細胞孔,輕輕混勻各管慢病毒稀釋液,取90 µl加入每孔細胞中,放入37℃的細胞培養箱中過夜培養;已知滴度的對照病毒加入1-5號稀釋液,待測樣品加入1號和2號稀釋液。第三天,去除含慢病毒的培養基,加入1 ml的完全培養基;4天後,抽提RNA準備做RT-qPCR。總RNA根據本領域習知手段進行提取。
反轉錄cDNA
a) 取一滅菌的無RNA酶的eppendorf管,每個樣本加入如下組分得到Mix 1。
b) 輕輕混勻後,在25℃下孵育10 min。
c) 在42℃下孵育60 min。
d) 在85℃下孵育5 min終止反應。所得到的cDNA保存在-20℃備用。
Real-time PCR 檢測:目的基因的檢測用引子序列(5’-3’):
參考基因的檢測用引子序列(5’-3’):
PCR體系中各組分的體積如下:
PCR反應條件:
結果:
組分 | 體積 |
RNA(1µg) | 2 µl |
5×VILO Reaction Mix | 4 µl |
10×SuperScript Enzyme Mix | 2 µl |
DEPC-treated water | 12 µl |
Total | 20 µl |
primer名稱 | 引子序列5’to 3’ |
WPRE-F | CGCTATGTGGATACGCTGCTTTA(SEQ ID NO: 3) |
WPRE-R | GCAACCAGGATTTATACAAGGAGGA(SEQ ID NO: 4) |
primer名稱 | 引子序列5’to 3’ |
GAPDH-F | GTCTCCTCTGACTTCAACAGCG(SEQ ID NO: 5) |
GAPDH-R | ACCACCCTGTTGCTGTAGCCAA(SEQ ID NO: 6) |
組分 | 體積 |
超純水 | 7.2 µl |
2×SYBR Mix | 10 µl |
上游引子(10 μM) | 0.4 µl |
下游引子(10 μM) | 0.4 µl |
模板 | 2 µl |
總體系 | 20 µl |
預變性 | 95℃ | 45 s | |
PCR循環 | 95℃ | 15 s | 40 cycles |
60℃ | 60 s | ||
融解曲線 | 60℃→95℃ |
慢病毒名稱 | 滴度(TU/ml) |
PGMLV-CMV-4IgB7-H3 (CD276)WT-PGK-puro | 2.04×106 |
PGMLV-CMV-4IgB7-H3 (CD276)MT (c.C1488T)-PGK-Blasticidin | 2.11×106 |
上表說明,轉染野生型基因的慢病毒和轉染突變編碼基因的慢病毒滴度基本相同,可以用於後續的體外效果比較的平行試驗。
實施例3 突變體和野生型HEK293T細胞體外效果的比較
一、 慢病毒感染目標細胞
第一天,以合適的比例接種目標細胞HEK293T(購自上海中科院細胞庫)於培養皿中。
第二天,感染前,病毒原液從-80℃冰箱取出後冰浴融化,用含5 μg/ml Polybrene的新鮮培養基按合適的MOI值稀釋病毒原液,吸除對照組和處理組原有的培養基,含有慢病毒陰性對照(NC-)稀釋液加到對照組細胞中,含有慢病毒液稀釋液加到處理組細胞中。
第三天,更換培養液,在感染16 h後將含有慢病毒的培養液更換成正常培養液。
第五天,選擇合適的真核抗性篩選細胞,藥物篩選兩輪,細胞基本穩定(4IgB7-H3 (CD276)WT穩定表現株puro全致死濃度1.5 μg/ml,維持濃度0.75 μg/ml;4IgB7-H3 (CD276)MT穩定表現株blast全致死濃度8 μg/ml,維持濃度4 μg/ml)。
二、流式染色驗證基因過表現效果
實驗材料:HEK293T細胞;培養基;血清(GIBICO);抗生素:青黴素和鏈黴素(Penicillin,Streptomycin);胰酶:0.25% Trypsin(GIBICO);高純度質體抽提套組(QIAGEN);流式抗體;
實驗儀器:CO2
培養箱(Thermo);熒光顯微鏡(Olympus);生物安全櫃(Thermo),BD FACSCalibur 流式細胞儀。
1、病毒感染:分別將過表現的慢病毒和慢病毒陰性對照包裝好並感染目的細胞,透過流式抗體染色方法檢測過表現載體的過表現效果。
2、透過流式抗體細胞染色檢測目的基因的表現
A. 細胞直接收在流式管裡,細胞吸除上層培養基,加入500 μl的PBS(1% BSA)清洗細胞,在1000 rpm下離心5 min,將液體直接倒出,留100 μl左右的PBS(1% BSA),沒有100 μl直接補足至100 μl,重新懸浮細胞;
B. 加入合適體積的熒光標記一抗到已經準備好的細胞懸浮液裡,混勻(不要用槍頭混勻,推動混勻),在4℃下避光孵育30-50 min;
C. 加入1 ml的PBS(1% BSA)清洗細胞,在1000 rpm在5 min,將液體直接倒出,留300 μl左右的PBS(1% BSA),沒有300 μl直接補足300 μl,重新懸浮細胞。
D. 流式細胞儀分析。
HEK293T細胞結果如圖4所示,經流式驗證,4IgB7-H3 (CD276)WT穩定表現株和4IgB7-H3 (CD276)MT穩定表現株與對照組相比較,表現量升高,有過表現效果,由此說明4IgB7-H3 (CD276)WT和 4IgB7-H3 (CD276)MT穩定表現株細胞構建成功。
三、細胞增殖測定
細胞:HEK293T,DMEM培養基 + 10% 胎牛血清(GIBICO);0.25% Trypsin(GIBICO);熒光顯微鏡(Olympus);生物安全櫃(Thermo);CO2
培養箱(Thermo);離心機(Thermo);CCK-8(Fluka);自動化酵素免疫分析儀(北京天石醫療用品製作所)。
1、第一天,準備4個96孔盤,在96孔盤中接種細胞懸浮液(2000細胞/每孔),每組細胞3個複孔。將培養盤放在培養箱中預培養(37℃,5% CO2
)。
2、暫態轉染後,取其中一個96孔盤,向每孔加入5 μL CCK8溶液(注意不要在孔中生成氣泡,它們會影響OD值的讀數)。其他3塊96孔盤在暫態轉染24 h、48 h、72 h小時後進行同樣的操作。
3、將培養盤在培養箱內孵育3小時。
4、用酵素免疫分析測讀儀測定在450 nm處的吸光度。
HEK293T細胞實驗結果和數據
0 H | 平均 | 24 H | 平均 | |||||
CON | 0.344 | 0.345 | 0.341 | 0.343 | 0.558 | 0.561 | 0.563 | 0.561 |
NC | 0.345 | 0.348 | 0.349 | 0.347 | 0.556 | 0.559 | 0.557 | 0.557 |
WT | 0.344 | 0.345 | 0.347 | 0.345 | 0.559 | 0.561 | 0.567 | 0.562 |
MT | 0.354 | 0.355 | 0.352 | 0.354 | 0.631 | 0.632 | 0.633 | 0.632 |
WT+MT | 0.353 | 0.354 | 0.356 | 0.354 | 0.595 | 0.591 | 0.593 | 0.593 |
48 H | 平均 | 72 H | 平均 | |||||
CON | 0.886 | 0.894 | 0.89 | 0.890 | 1.019 | 1.023 | 1.018 | 1.020 |
NC | 0.891 | 0.892 | 0.889 | 0.891 | 1.021 | 1.025 | 1.026 | 1.024 |
WT | 0.934 | 0.931 | 0.932 | 0.932 | 1.131 | 1.132 | 1.133 | 1.132 |
MT | 0.996 | 0.992 | 0.998 | 0.995 | 1.235 | 1.234 | 1.236 | 1.235 |
WT+MT | 0.958 | 0.956 | 0.948 | 0.954 | 1.208 | 1.211 | 1.205 | 1.208 |
其中,CON代表空細胞,NC代表感染野生型對照慢病毒和突變型對照慢病毒的細胞株,WT代表感染4IgB7-H3 (CD276)WT野生型慢病毒的細胞株,MT代表感染4IgB7-H3 (CD276)MT突變型慢病毒的細胞株,WT+MT代表同時感染4IgB7-H3 (CD276)WT野生型慢病毒和4IgB7-H3 (CD276)MT突變型慢病毒的細胞株。
上表數據的統計學分析結果如下:
WT和MT變異數分析 | |||||||
差異源 | SS | df | MS | F | P-value | F crit | |
組間 | 0.015914 | 1 | 0.015914 | 15913.5 | 2.37E-08 | 21.19769 | |
組內 | 4E-06 | 4 | 0.000001 | ||||
總計 | 0.015918 | 5 | |||||
上表中,SS代表離均差平方和,df代表自由度。MS代表均方,F代表F統計量,P-value代表p值,F crit代表F臨界值。
圖5進一步顯示了上表的細胞增殖趨勢。
結論:上述細胞株培養72 h後經CCK8檢測驗證,4IgB7-H3 (CD276)WT穩定表現株、4IgB7-H3 (CD276)MT穩定表現株和同時感染4IgB7-H3 (CD276)WT野生型表現慢病毒和4IgB7-H3 (CD276)MT突變型表現慢病毒的細胞株與對照細胞相比,細胞增殖能力增强;4IgB7-H3 (CD276)WT穩定表現株與4IgB7-H3 (CD276)MT穩定表現株相比,p=2.37E-8<0.01有顯著統計學意義。由此可見,轉染了人4IgB7-H3的突變編碼基因的HEK293T細胞比轉染了4IgB7-H3野生型基因的細胞增殖快,說明突變編碼基因在HEK293T細胞中比野生型基因具有更强的例如促進增殖的生物學功能。
實施例4 突變體和野生型jurkat細胞體外效果的比較
一、慢病毒感染目標細胞
本實施例中,慢病毒感染的目標細胞爲jurkat細胞(購自上海中科院細胞庫),感染方法同實施例3。至第五天選擇合適的真核抗性篩選細胞,藥物篩選兩輪,細胞基本穩定。在jurkat細胞中,4IgB7-H3 (CD276) WT穩定表現株puro全致死濃度1.5 μg/ml,維持濃度0.75 μg/ml;4IgB7-H3 (CD276) MT穩定表現株blast全致死濃度7 μg/ml,維持濃度3.5 μg/ml。
二、流式染色驗證基因過表現效果
流式染色驗證基因過表現效果時,除了細胞採用jurkat細胞以外,其它條件同實施例3。
jurkat細胞中結果如圖6所示,單獨感染4IgB7-H3 (CD276)WT穩定表現株和4IgB7-H3 (CD276)MT穩定表現株與對照組相比較,表現量升高,有過表現效果。同時感染4IgB7-H3 (CD276)WT野生型表現慢病毒和4IgB7-H3 (CD276) MT突變型表現慢病毒的細胞株(4IgB7-H3 (CD276) WT+MT)和對照組相比較,表現量升高,也有過表現效果,由此說明4IgB7-H3 (CD276) WT、4IgB7-H3 (CD276) MT穩定表現株以及4IgB7-H3 (CD276) WT+MT穩定表現株細胞構建成功。
以下爲圖6中的縮略語說明:
A,jurkat不染:jurkat空細胞,無熒光標記;
B,jurkat:jurkat空細胞;
C,NC染:PGK-Puro+ PGK-Blasticidin,感染野生型對照慢病毒和突變型對照慢病毒的jurkat細胞;
D,4IgB7-H3 (CD276)WT:單獨感染4IgB7-H3 (CD276)WT野生型表現慢病毒的jurkat細胞;
E,4IgB7-H3 (CD276)MT:單獨感染4IgB7-H3 (CD276)MT突變型表現慢病毒的jurkat細胞;
F,4IgB7-H3 (CD276)WT+MT:同時感染4IgB7-H3 (CD276)WT野生型表現慢病毒和4IgB7-H3 (CD276)MT突變型表現慢病毒的jurkat細胞;
jurkat IgG:同型對照;
WT-IgG:同型對照。
三、細胞增殖測定
同樣地,除了細胞採用jurkat細胞以外,其它條件同實施例3對應的實驗。
jurkat細胞實驗結果和數據見下表及圖7:
0 H | 平均 | 24 H | 平均 | |||||
CON | 0.243 | 0.239 | 0.24 | 0.241 | 0.429 | 0.439 | 0.444 | 0.437 |
NC | 0.246 | 0.251 | 0.242 | 0.246 | 0.447 | 0.442 | 0.445 | 0.445 |
WT | 0.269 | 0.272 | 0.272 | 0.271 | 0.456 | 0.457 | 0.461 | 0.458 |
MT | 0.244 | 0.241 | 0.246 | 0.244 | 0.432 | 0.423 | 0.445 | 0.433 |
WT+MT | 0.233 | 0.241 | 0.239 | 0.238 | 0.453 | 0.457 | 0.464 | 0.458 |
48 H | 平均 | 72 H | 平均 | |||||
CON | 0.752 | 0.754 | 0.754 | 0.753 | 1.140 | 1.129 | 1.205 | 1.158 |
NC | 0.747 | 0.743 | 0.746 | 0.745 | 1.115 | 1.123 | 1.115 | 1.118 |
WT | 0.721 | 0.723 | 0.73 | 0.725 | 1.211 | 1.199 | 1.210 | 1.207 |
MT | 0.695 | 0.693 | 0.704 | 0.697 | 0.989 | 0.997 | 0.999 | 0.995 |
WT+MT | 0.71 | 0.707 | 0.71 | 0.709 | 1.002 | 0.997 | 0.947 | 0.982 |
圖7中,jurkat指未轉染病毒的空細胞,NC指感染野生型對照慢病毒和突變型對照慢病毒的jurkat細胞株,WT爲感染4IgB7-H3 (CD276)WT野生型表現慢病毒的jurkat細胞株,MT爲感染4IgB7-H3 (CD276)MT突變型表現慢病毒的jurkat細胞株,WT+MT(或稱雜合型)爲同時感染4IgB7-H3 (CD276)WT野生型表現慢病毒和4IgB7-H3 (CD276)MT突變型表現慢病毒的jurkat細胞株。
上表數據的統計學分析結果如下:
WT和MT變異數分析 | |||||||
差異源 | SS | df | MS | F | P-value | F crit | |
組間 | 0.075713 | 1 | 0.075713 | 156.216 | 0.000236 | 7.708647 | |
組內 | 0.001939 | 4 | 0.000485 | ||||
總計 | 0.077651 | 5 | |||||
上表中,SS代表離均差平方和,df代表自由度。MS代表均方,F代表F統計量,P-value代表p值,F crit代表F臨界值。
圖7的結果表明,上述細胞株培養72 h後經CCK8檢測驗證,WT+MT(即雜合型)穩定表現株與jurkat細胞以及WT細胞相比較,WT+MT(即雜合型)穩定表現株的細胞增殖減慢,由此可以表明雜合型對T細胞增殖的抑制性增强;WT+MT穩定表現株與WT穩定表現株相比,p=0.000236<0.01有顯著統計學意義。
jurkat細胞屬人急性T淋巴細胞白血病細胞株,可模擬T淋巴細胞功能,在T淋巴細胞信號轉導、細胞激素和受體表現的體外研究中應用廣泛,其體外實驗結果揭示突變編碼4IgB7-H3與抑制性受體結合對T淋巴細胞的增殖的抑制作用增强。由此可推知,本發明的如SEQ ID NO: 1所示的新的人4IgB7-H3的突變編碼基因在T、B細胞增殖、活化、輔助T細胞分化,信號轉導及作用細胞的細胞激素分泌過程中產生重要作用,對腫瘤逃避免疫監視中難以誘導腫瘤特異性T細胞活化的主要原因產生了解決及治療作用。其在製備基因組工程、基因重組、以T細胞和樹突狀細胞爲基礎的治療惡性腫瘤和傳染性疾病的免疫治療藥物、疫苗以及蛋白替補療法、緩解過敏等中有突出而廣泛的應用前景。
無
圖1爲構建的含人4IgB7-H3的突變編碼基因的重組表現載體的質體圖譜;
圖2爲構建的含編碼人4IgB7-H3的野生型基因的重組表現載體的質體圖譜;
圖3爲重組表現載體的流式細胞檢測結果:A. 含人4IgB7-H3的突變編碼基因的重組表現載體,B. 含編碼人4IgB7-H3的野生型基因的重組表現載體;
圖4爲慢病毒感染HEK293T細胞的流式細胞檢測結果:A. 含人4IgB7-H3的突變編碼基因的慢病毒,B. 含編碼人4IgB7-H3的野生型基因的慢病毒;
圖5爲野生型和突變編碼基因對HEK293T細胞增殖效果隨時間變化影響的比較;
圖6爲慢病毒感染jurkat細胞的流式細胞檢測結果;
圖7爲野生型和突變編碼基因對jurkat細胞增殖效果隨時間變化影響的比較。
無
Claims (10)
- 一種人4IgB7-H3的突變編碼基因,其特徵在於,所述人4IgB7-H3的突變編碼基因的核苷酸序列如SEQ ID NO: 1所示。
- 一種如請求項1所述的突變編碼基因編碼的蛋白或其受體或配體;較佳地,所述蛋白的第一個C樣結構域末端包含PQRSPT模體。
- 一種重組表現載體,其特徵在於,所述重組表現載體包含如請求項1所述的人4IgB7-H3突變編碼基因;較佳地,所述重組表現載體爲病毒表現載體或原核表現載體或真核表現載體;更佳地,所述病毒表現載體爲慢病毒表現載體或腺相關病毒表現載體或反轉錄病毒載體或腺病毒載體或單純孢疹病毒。
- 一種宿主細胞,其特徵在於,所述宿主細胞包括如請求項3所述的重組表現載體;較佳地,所述宿主細胞爲真核細胞,更佳為哺乳動物細胞。
- 一種分離的mRNA,其特徵在於,所述mRNA由如SEQ ID NO: 1所示的核苷酸序列轉錄而得;較佳地,所述mRNA爲IVT mRNA。
- 一種細胞,包含如請求項5所述的分離的mRNA和RNA感應器和/或mRNA免疫識別結構元件;較佳地,所述細胞包括免疫細胞和非免疫細胞;更佳地所述免疫細胞包括樹突狀細胞和淋巴細胞,所述淋巴細胞包括B細胞和T細胞,所述T細胞包括輔助性T細胞、抑制性T細胞、作用T細胞、細胞毒性T細胞、遲發性變態反應T細胞、天然T細胞和記憶T細胞;其中,輔助性T細胞分爲Th1、Th2、Th17或Treg。
- 一種分離的miRNA,其特徵在於,所述分離的miRNA標靶SEQ ID NO: 1和/或SEQ ID NO: 2所示的核苷酸;較佳地,所述分離的miRNA標靶SEQ ID NO: 1和/或SEQ ID NO: 2所示的核苷酸的第2142個位點。
- 一種標靶藥物,其特徵在於,所述標靶藥物標靶如請求項1所述的人4IgB7-H3的突變編碼基因,所述標靶藥物爲反義RNA、siRNA、環狀RNA、lncRNA或shRNA;或者標靶如請求項2所述的蛋白或其受體或配體,所述標靶藥物例如爲抗體。
- 一種藥物組合物,其特徵在於,其包括如請求項1所述的人4IgB7-H3突變編碼基因、如請求項2所述的蛋白或其受體或配體、如請求項3所述的重組載體、如請求項4所述的宿主細胞、如請求項5所述的分離的mRNA、如請求項6所述的細胞、如請求項7所述的分離的miRNA或如請求項8所述的標靶藥物;較佳地,所述藥物化合物包括藥學上可接受的載體或賦形劑。
- 一種如請求項1所述的人4IgB7-H3的突變編碼基因、如請求項2所述的蛋白或其受體或配體、如請求項3所述的重組載體、如請求項4所述的宿主細胞、如請求項5所述的分離的mRNA、如請求項6所述的細胞、如請求項7所述的分離的miRNA、如請求項8所述的標靶藥物或如請求項9所述的藥物組合物在製備調節T細胞活化和/或調節T細胞增殖的藥物中的用途; 進一步地,在製備調節免疫或免疫治療的藥物中的用途;較佳地,所述調節免疫或免疫治療的藥物爲預防和/或治療腫瘤、傳染性疾病或緩解過敏的藥物;更佳地,所述腫瘤較佳爲胃癌、腸癌、肝癌、肺癌、膀胱癌、睪丸癌、前列腺癌、子宮頸癌、乳房癌和淋巴癌中的一種或多種; 所述藥物較佳爲疫苗。
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