pRetro-on 载体

pRetro-On is a Moloney murine leukemia virus (MoMuLV)-derived retroviral vector expressing the reverse tetracycline-controlled transactivator (rtTA) from the SV40 promoter. rtTA is a fusion of amino acids 1-207 of the tet repressor (rTetR) and the negatively charged C-terminal activation domain (130 amino acids) of the VP16 protein of herpes simplex virus. The tet-responsive element (TRE) controls expression of the gene of interest cloned into the MCS. With the pRetro-On Vector, rtTA binds the TRE and activates transcription in the presence of the Tc derivative doxycycline (Dox). As Dox is removed from the culture medium, transcription from the TRE is turned off in a highly dose-dependent manner. rtTA and TRE are derived from vectors described previously. The 5' viral LTR controls expression of the transcript that contains Psi+ (the extended viral packaging signal), and the puromycin resistance (Puror) gene for antibiotic selection in mammalian cells. It is not necessary to include a polyadenylation signal in the sequence cloned into the MCS. pRetro-On also includes the E. coli Ampr gene for antibiotic selection in E. coli. pRetro-On can be distinguished from pRetro-Off by digestion with Hind III and BamH I. (pRetro-Off should produce fragments of 1.2 and 5.8 kb, whereas pRetro-On should produce fragments of 0.9 and 5.9 kb.) pRetro-On can be transfected into a high titer packaging cell line and thereby mediate production of infectious, replication-incompetent retroviral particles. The pRetro-On retroviral genome lacks the viral genes gag, pol, and env, which are supplied by the packaging cell line. The transcript produced by the pRetro-On construct is recognized by the viral structural proteins expressed in a packaging cell line and packaged into infectious retroviral particles. Because the RNA transcript packaged in these particles does not contain the viral genes, it cannot replicate in the target cells that it infects. The level of induction in cell populations infected with this vector depends on the efficiency of infection and on the titer of the virus. Viral supernatants with titers >105 cfu/ml should be produced to achieve high-level induction.

pRetro-on 载体原理机制

载体效应实验 用牛血清白蛋白耦联二硝基苯 (BSA-DNP) 和卵白蛋白耦联二硝基苯 (OA-DNP) 制备载体—半抗原，观察载体在抗体产生中的作用。首先以 BSA － DNP 对小鼠进行初次免疫，然后分别用 BSA － DNP 和 OA － DNP 进行再次免疫，观察载体对抗 DNP 抗体产生的影响。结果发现，只有当初次和再次免疫时，半抗原耦联在相同的载体上才能产生抗半抗原抗体的再次应答。此作用被称为载体效应。载体效应实验证明了载体不是单纯起运载半抗原的作用，而是具有载体特异性。因此揭示了一个完全抗原分子，必须具有pRetro-on 载体决定基和半抗原决定基。并进一步证明了在抗体形成过程中存在有对载体特异性反应细胞，即 T 淋巴细胞为载体的反应细胞，对抗体产生起辅助作用；也存在对半抗原特异性反应细胞，即 B 淋巴细胞。

实验组 初次免疫 再次免疫 抗DNP抗体

1 BSA-DNP BSA-DNP +++

2 BSA-DNP OA-DNP +

3 BSA-DNP+OA OA-DNP +++

抗体的特异性来自于B细胞膜表面Ig受体对抗原的特异性识别，以致B细胞只能和其特异性识别的抗原结合并进行加工处理。但是作为T细胞依赖性抗原（Td-Ag），被B细胞或巨噬细胞加工后，只剩下一些MHC-Ⅱ类分子结合的抗原寡肽段被呈递给Th细胞，以产生反馈性的活化该特异性B细胞克隆的信号，并使B细胞分化成抗体分泌细胞。 [1]

在一个完整的大抗原分子上与Ab结合的表位和与MHC-TCR结合的序列可能不同，与抗体结合的表位相当于半抗原部分与MHC-TCR结合序列相当于半抗原载体部分。从而显示出在Td-Ag中半抗原与载体效应发生的分子基础。

同样的半抗原与不同的载体偶联结合都可以产生相应Ab，但其辅助T细胞可以不用。特异性的抗体产生后，可引起一系列的免疫效应。

在免疫应答中，B细胞识别半抗原，并提呈载体表位给CD4阳性T细胞；Th细胞识别载体表位。故通过载体把特异的T-B细胞之间连接起来，T细胞才能激活B细胞，使B细胞分泌抗体。这就是载体效应。