共刺激分子CD137L对HBsAg重组疫苗诱导小鼠免疫应答的佐剂作用

发布时间：2018-03-21 16:33

  本文选题：CD137L　切入点：共刺激分子　出处：《杭州师范大学》2012年硕士论文　论文类型：学位论文

【摘要】：乙型肝炎病毒(Hepatitis B virus, HBV)持续感染的主要机理之一是特异性细胞免疫应答不足,而诱导有效细胞免疫的治疗性疫苗有望增强抗病毒免疫,可作为HBV感染综合治疗的有效手段之一。因此,有效性、持续性和安全性兼顾的治疗性疫苗是慢性乙肝免疫治疗研究的一个重要目标。 目前市售的含铝佐剂乙肝表面抗原(hepatitis B surface antigen, HBsAg)重组疫苗是有效的预防用疫苗,但由于诱导的细胞免疫应答不足,因而一般不用于抗病毒治疗。 CD137/CD137L (4-1BB/4-1BBL)是一对重要的协同刺激信号分子,对增强和维持免疫应答,尤其是特异性T细胞的扩增、存活和记忆性杀伤等均有极其重要的作用,在抗病毒免疫治疗中具有潜在的临床应用价值。 本研究构建小鼠CD137L的真核表达质粒,观察该重组质粒和CD137L重组蛋白在小鼠体内对HBsAg重组蛋白和含铝佐剂乙肝疫苗诱导特异性免疫应答的免疫调节作用,探讨CD137L分子作为乙肝重组疫苗的佐剂以增强抗病毒免疫效应的可能性,为慢性乙型肝炎的治疗性疫苗提供新的优化思路。 第一部分：小鼠CD137L真核表达质粒的构建及鉴定目的 构建小鼠CD137L的真核表达质粒,并将该质粒体外转染NIH3T3细胞,检测其CD137L mRNA和蛋白的表达,为进一步用于动物实验做好前期准备。方法 1.重组质粒pcD137L的构建 分离BALB/c小鼠脾细胞,体外经ConA刺激培养后提取细胞总RNA,通过RT-PCR克隆小鼠CD137L全长cDNA, PCR产物经回收和纯化后克隆到载体pcDNA3.1(+)上。重组质粒分别经PCR、双酶切和测序鉴定。 2.重组质粒pcD137L的表达及检测 以脂质体法将重组质粒pcD137L转染到NIH3T3细胞中,并通过RT-PCR、流式细胞术和免疫荧光法分别检测转染细胞中CD137L mRNA和蛋白的表达。结果 1.重组质粒pcD137L的PCR产物扩增出与目的片段相符的单一条带；双酶切鉴定得到与目的基因大小一致的片段。 2.测序结果证实所插入片段的核苷酸序列与基因库中小鼠CD137L基因序列完全一致。 3.经RT-PCR从pcD137L转染细胞中扩增出与目的基因相符的单一条带。 4. pcD137L转染细胞经PE-抗小鼠CD137L单抗染色后,流式细胞仪检测到其表面CD137L分子的表达,并在荧光显微镜下观察到这些细胞的胞膜呈现荧光。结论 1.成功构建了小鼠CD137L的真核表达载体(pcD137L). 2.证实重组质粒pcD137L在真核细胞内能成功表达CD137L mRNA和目的蛋白。 第二部分：CD137L重组质粒和CD137L重组蛋白对HBsAg重组疫苗的佐剂作用 目的 分别以CD137L重组质粒(pcD137L)和重组蛋白(rCD137L)与HBsAg重组疫苗共免疫小鼠,探讨CD137L分子作为重组乙肝表面抗原(rHBsAg)疫苗的佐剂以增强抗病毒免疫治疗作用的可能性。 方法 1.动物分组和免疫： BALB/c小鼠随机分8个实验组和5个对照组,每组5只。在0,3,6周各免疫1次,共3次,最后1次免疫后1周处死小鼠。 实验组为：(1) rHBsAg组(S);(2) rHBsAg+pcDNA3.1(+)组(S+pcD);(3)rHBsAg+pcD137L组(S+pcD137L);(4)rHBsAg+铝佐剂组(S+Al);(5)rHBsAg+铝佐剂+pcDNA3.1(+)组(S+Al+pcD);(6) rHBsAg+铅佐剂+pcD137L组(S+Al+pcD137L);(7) rHBsAg+rCD137L组(S+rCD137L);(8) rHBsAg+铝佐剂+rCD137L组(S+Al+rCD137LX 对照组为：(1)NS组；(2) pcDNA3.1(+)组(pcD)；(3)铝佐剂组(A1)；(4) pcD137L组；(5) rCD137L组。 2. ELISA法检测免疫后小鼠血清IgG, IgGl及IgG2a水平。 3.流式细胞术检测脾细胞中CD8+T细胞内IFN-y/IL-4表达水平。 4.流式细胞术分析脾细胞中CD44+high釉CD127+CD44+highCD8+T记忆细胞百分比。 5.流式细胞术分析特异性CTL细胞诱导靶细胞凋亡的活性。 结果 1.血清特异性抗体抗-HBs水平检测 (DpcD137L能促进rHBsAg诱导抗体应答(总IgG、IgG2a和IgGl增高),对含铝佐剂乙肝疫苗抗体应答无调节作用。 (2) rCD137L增强rHBsAg诱导Thl型抗体应答(总IgG和IgG2a增高),并可增强含铝佐剂乙肝疫苗诱导Thl型抗体而下调Th2型抗体(上调IgG2a,下调IgGl). (3)铝佐剂对rHBsAg诱导抗体有显著增强作用(总IgG、IgG2a和IgGl增高)。 2. CD8+T细胞内IFN-y/IL-4检测 (1) pcD137L可促进rHBsAg诱导特异性CD8+T细胞IFN-y的产生(Tcl),并可增强含铝佐剂乙肝疫苗诱导Tcl反应。 (2) rCD137L对rHBsAg诱导Tcl细胞应答无明显作用,但对含铝佐剂乙肝疫苗诱导Tcl细胞免疫应答有显著促进作用。 (3)铝佐剂可增强rHBsAg的免疫原性,提高Tcl细胞免疫应答水平。 (4) pcD137L和rCD137L对rHBsAg或含铝佐剂rHBsAg刺激特异性CD8+T细胞分泌IL-4均无明显调节作用。 3.脾细胞CD8+记忆T细胞分析 (1) pcD137L促进rHBsAg产生CD44+high和CD127+CD44+highCD8+T记忆细胞,但对含铝佐剂乙肝疫苗诱导CD8+记忆T细胞无明显作用。 (2) rCD137L可促进rHBsAg诱导CD8+记忆T细胞形成,并可增强含铝佐剂乙肝疫苗产生CD8+记忆T细胞。 (3)铝佐剂促进rHBsAg疫苗诱导CD8+记忆T细胞数。 4.特异性CTL诱导靶细胞凋亡的活性分析 (1) rCD137L增强rHBsAg及含铝佐剂rHBsAg诱导特异性CTL应答。 (2)铝佐剂可增强rHBsAg疫苗的CTL活性。 以上差异均有统计学意义。结论 1. CD137L重组质粒增强HBsAg重组疫苗在小鼠体内诱导的抗体应答、Tcl细胞应答及CD8+记忆T细胞形成,但对含铝佐剂乙肝疫苗无明显免疫增强作用。 2.CD137L重组蛋白显著促进含铝佐剂HBsAg重组疫苗在小鼠体内产生IgG2a (Thl型)抗体反应和以分泌IFN-y为特征的Tc1(I型)细胞免疫,增强其特异性CTL活性及CD8+记忆T细胞形成,可能是市售乙肝疫苗由预防用制剂转为治疗性疫苗的有效佐剂。
[Abstract]:Hepatitis B virus (Hepatitis B, virus, HBV) is one of the main mechanism of persistent infection is the specific cellular immune response and induce cellular immune deficiency, effective therapeutic vaccine could enhance antiviral immunity, can be used as one of the effective means of comprehensive treatment of HBV infection. Therefore, the validity, continuity and security of both therapeutic vaccine is an important target on chronic hepatitis B immune therapy.
Currently, the commercially available recombinant hepatitis B surface antigen (HBsAg) recombinant vaccine is an effective preventive vaccine, but it is generally not used for antiviral therapy because of its insufficient cellular immune response.
CD137/CD137L (4-1BB/4-1BBL) is one of the important costimulatory signal molecule, maintain and enhance immune response, especially the amplification of specific T cell and the effect of survival and mnemonic killer has the extremely important and has potential clinical value in antiviral therapy.
The construction of eukaryotic expression plasmid of CD137L mice, observe the immune regulation effect of recombinant plasmid and CD137L recombinant protein in mice by recombinant HBsAg protein and aluminum adjuvant hepatitis B vaccine induced specific immune response, the possibility of CD137L as a molecular adjuvant of recombinant HB vaccine to enhance the antiviral immune effect, provide new optimization ideas for the treatment of chronic hepatitis B vaccine.
Part 1: Construction and identification of CD137L eukaryotic expression plasmids in mice
The eukaryotic expression plasmid of mouse CD137L was constructed, and the plasmid was transfected into NIH3T3 cells in vitro, and the expression of CD137L mRNA and protein was detected.
Construction of 1. recombinant plasmid pcD137L
The spleen cells from BALB/c mice were isolated, cultured in vitro, and the total RNA was extracted after ConA stimulation. The full length cDNA of mouse CD137L was cloned by RT-PCR, and PCR products were cloned into vector pcDNA3.1 (+) by recycling and purification. The recombinant plasmids were identified by PCR, double enzyme digestion and sequencing.
Expression and detection of 2. recombinant plasmid pcD137L
The recombinant plasmid pcD137L was transfected into NIH3T3 cells by liposome method, and the expression of CD137L mRNA and protein in transfected cells was detected by RT-PCR, flow cytometry and immunofluorescence.
1. the PCR product of recombinant plasmid pcD137L amplified a single band that was consistent with the target fragment, and double enzyme digestion was used to identify the fragments that were in accordance with the size of the target genes.
The results of 2. sequencing confirmed that the nucleotide sequence of the inserted fragment was exactly the same as the mouse CD137L gene sequence in the gene pool.
3. the single band that was consistent with the target gene was amplified by RT-PCR from the transfected cells from pcD137L.
4. pcD137L transfected cells were stained with PE- anti mouse CD137L monoclonal antibody. The expression of CD137L on their surface was detected by flow cytometry, and the fluorescence of these cells was observed under fluorescence microscope.
1. the eukaryotic expression vector (pcD137L) of mouse CD137L was successfully constructed.
2. it was confirmed that the recombinant plasmid pcD137L could successfully express CD137L mRNA and target protein in eukaryotic cells.
The second part: the effect of CD137L recombinant plasmid and CD137L recombinant protein on the adjuvant of HBsAg recombinant vaccine
objective
Mice were immunized with recombinant plasmid CD137L (pcD137L) and recombinant protein (rCD137L) and recombinant HBsAg vaccine respectively. The possibility of CD137L as a adjuvant of recombinant hepatitis B surface antigen (rHBsAg) vaccine was explored to enhance the effect of antiviral immunotherapy.
Method
1. animal groups and immunization:
BALB/c mice were randomly divided into 8 experimental groups and 5 control groups, with 5 rats in each group. 1 times each were immunized at 0,3,6 weeks, and the mice were killed for 1 weeks after the last 1 immunizations.
The experimental group: (1) rHBsAg group (S); (2) rHBsAg+pcDNA3.1 (+) group (S+pcD); (3) rHBsAg+pcD137L group (S+pcD137L); (4) rHBsAg+ aluminum adjuvant group (S+Al); (5) rHBsAg+ aluminum adjuvant +pcDNA3.1 (+) group (S+Al+pcD); (6) rHBsAg+ lead adjuvant + pcD137L group (S+Al+pcD137L); (7) rHBsAg+rCD137L group (S+rCD137L); (8) +rCD137L group (S+Al+rCD137LX rHBsAg+ aluminum adjuvant
The control group was: (1) NS group; (2) pcDNA3.1 (+) group (pcD); (3) aluminum adjuvant group (A1); (4) pcD137L group; (5) rCD137L group.
The serum levels of IgG, IgGl and IgG2a after immunization were detected by 2. ELISA method.
The expression of IFN-y/IL-4 in CD8+T cells in spleen cells was detected by 3. flow cytometry.
4. flow cytometry was used to analyze the percentage of CD44+high enamel CD127+CD44+highCD8+T memory cells in splenocytes.
5. flow cytometry was used to analyze the activity of specific CTL cells to induce the apoptosis of the target cells.
Result
Detection of anti -HBs level of 1. serum specific antibodies
(DpcD137L can promote rHBsAg induced antibody response (the increase of total IgG, IgG2a and IgGl), and has no regulatory effect on the antibody response of HBV vaccine containing aluminum adjuvant.
(2) rCD137L enhanced rHBsAg induced Thl type antibody response (increased total IgG and IgG2a), and increased the Thl antibody induced by hepatitis B vaccine with aluminum adjuvant, and down regulated Th2 antibody (up IgG2a and down IgGl).
(3) the aluminum adjuvant enhanced the antibody induced by rHBsAg (total IgG, IgG2a and IgGl).
Detection of IFN-y/IL-4 in 2. CD8+T cells
(1) pcD137L can induce rHBsAg to induce the production of IFN-y in specific CD8+T cells (Tcl), and enhance the Tcl reaction induced by the hepatitis B vaccine containing aluminum adjuvant.
(2) rCD137L has no obvious effect on rHBsAg induced Tcl cell response, but it can significantly promote the immune response of Tcl cells induced by hepatitis B vaccine containing aluminum adjuvant.
(3) aluminum adjuvant can enhance the immunogenicity of rHBsAg and improve the level of Tcl cell immune response.
(4) both pcD137L and rCD137L had no obvious regulatory effect on rHBsAg or aluminum containing adjuvant rHBsAg stimulated by specific CD8+T cells to secrete IL-4.
Analysis of CD8+ memory T cells in 3. splenocytes
(1) pcD137L promoted rHBsAg to produce CD44+high and CD127+CD44+highCD8+T memory cells, but had no obvious effect on CD8+ memory T cells induced by the hepatitis B vaccine containing aluminum adjuvant.
(2) rCD137L can induce rHBsAg to induce the formation of CD8+ memory T cells and enhance the CD8+ memory T cells produced by the vaccine of hepatitis B containing adjuvant hepatitis B.
(3) aluminum adjuvant promoted the rHBsAg vaccine to induce the number of CD8+ memory T cells.
Analysis of the activity of 4. specific CTL induced apoptosis in target cells
(1) rCD137L enhanced rHBsAg and aluminum containing adjuvant rHBsAg induced specific CTL responses.
(2) the aluminum adjuvant can enhance the CTL activity of rHBsAg vaccine.
The above differences were statistically significant.
1. CD137L recombinant plasmid enhanced the antibody response induced by HBsAg recombinant vaccine in mice, Tcl cell response and CD8+ memory T cell formation, but had no significant immune enhancing effect on hepatitis B vaccine containing aluminum adjuvant.
The recombinant 2.CD137L protein significantly promote aluminum adjuvant HBsAg recombinant vaccine IgG2a in mice (Thl) antibody reaction and to secrete IFN-y as the characteristics of the Tc1 (I) cell immunity, enhance the specific activity of CTL and CD8+ T memory cell formation, may be sold by the prevention of hepatitis B vaccine preparations for effective adjuvant therapy vaccines.

【学位授予单位】：杭州师范大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392.11

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1 江虹;黄茵;史丽云;吴炜;蔡玲斐;贾红宇;钟石根;;CD137L在HBsAg DNA疫苗诱导小鼠细胞免疫应答中的佐剂效应[J];浙江大学学报(医学版);2010年04期

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