mRNA疫苗因其特有优势在新型冠状病毒肺炎疫情爆发期间备受关注。它利用人体细胞来产生病毒抗原蛋白，从而激发免疫应答。与传统疫苗相比，mRNA疫苗诸多优势，但其不稳定性也会大大降低翻译效率。5′UTR是决定mRNA疫苗表达效率的重要结构，其二级结构影响核糖体的募集和起始密码子的扫描。研究人员通过优化5′UTR序列来提升mRNA的翻译效率。合理设计高效蛋白表达的5′UTR仍具有很大挑战性，在已知的序列里寻找表达能力强的5'UTR也是一种好的方法。由于mRNA翻译效率受5′UTR、3′UTR共同调节较为复杂，且本实验研究对象为5′UTR，因此本实验所有mRNA均没有3′UTR。

单纯疱疹病毒1型（HSV-1）隶属α-疱疹病毒科，会引起口腔、唇部、眼角膜等面部的病变，无法彻底治愈进而严重影响患者的生活质量。HSV-1入侵宿主细胞后，病毒基因组内各基因受病毒本身级联调控有序的表达。感染1小时后，五个即刻早期基因最先迅速表达，这些即刻早期基因上游具有相似的序列。通过查阅大量文献及对比序列，我们发现即刻早期基因上游非编码序列前半部分存在顺式作用模块及大量元件，后半部分是经过Genbank可查到单纯疱疹病毒1型17毒株基因序列（NC_001806.2）mRNA序列得出的5′UTR序列。有研究通过敲除即刻早期基因RL2、RS1、US1后发现其早期基因、晚期基因在感染1.5小时的表达水平异常增高，病毒蛋白在表达时序上的不同似乎是由这些特殊的上游非编码序列所决定，由此可以体现出HSV-1 即刻早期基因的非编码序列的重要性。

基于这些研究提出了一个问题：即刻早期基因的上游非编码序列的两部分分别作为模式蛋白mRNA的5′UTR对其表达有怎样的影响。为了解决这个问题，本实验第一部分将增强绿色荧光蛋白作为模式蛋白用于检测即刻早期基因RL2、RS1上游非编码序列前半部分作为其mRNA的5′UTR对表达水平的影响，结果显示模式蛋白完全不表达。考虑到RL2和RS1的上游非编码序列分布顺式作用模块且每个模块都分布着大量元件，我们将WTA、WTD、WTE三个顺式作用模块作为模式蛋白mRNA的5′UTR，发现模式蛋白的同样不表达。在此基础上根据三个顺式作用模块序列的元件分布依次删减元件得到了13条序列，将13条序列作为模式蛋白mRNA的5′UTR，其中WTA1、WTA2、ASP1、DSP1、Degr、KLF 6条序列均有不同水平的模式蛋白表达，具有一定的实用价值。实验第一部分得出即刻早期基因的上游非编码序列前半部分不具有作为mRNA疫苗的5′UTR能力的结论，但感染初期即刻早期基因表达水平迅速升高是毋庸置疑的。考虑到上游非编码序列前半部分和编码序列之间的是即刻早期基因自身具有的5′UTR，这些5′UTR是否是表达水平好的mRNA 5′UTR值得我们探究。实验第二部分我们将RL2、RS1、US1、US12的5′UTR作为模式蛋白mRNA的5′UTR，利用mRNA转染试剂和纳米脂质体颗粒两种递送方法分别在体外细胞和动物体内检验了模式蛋白的表达水平差异。其中RL2的5′UTR表达水平与对照β珠蛋白5′UTR接近，RS1的5′UTR表达水平稍弱于对照5′UTR，RL2的5′UTR有潜力成为mRNA疫苗的5′UTR。我们对RS1、US1、US12 5′UTR表达能力表达能力弱于RL2的5′UTR进行了分析。一方面，具有RL2 5′UTR的EGFP mRNA稳定性更好；另一方面，我们通过序列比对发现RS1、US1、US12的5′UTR上重复出现gcatcg序列。

gcatcg序列多次出现在即刻早期基因非编码序列引起了我们的兴趣，这段序列的存在可能与即刻早期基因的快速表达有关。实验第三部分从靠近起始密码子位置的gcatcg依次删减，观察依次删减gcatcg对模式蛋白表达的影响。结果显示删减掉第一个gcatcg后的序列能够使模式蛋白的水平与β珠蛋白的5′UTR相近，删减了两个gcatcg后模式蛋白完全不表达。根据这一结果我们大胆猜测gcatcg或包含gcatcg的序列可能一个未发现的潜在元件，即刻早期基因5′UTR序列中存在一个gcatcg有利于mRNA表达，而两个gcatcg的存在却能降低mRNA表达水平，但此结果仍需要进一步验证。

5′UTR序列对于mRNA疗法和疫苗研发均具有重要意义。虽然目前在5′UTR上发现了少量类似Kozak序列的元件，但在提高外源蛋白表达方面并没有太大进展。本研究初步证明RL2和RS1的上游非编码序列前半部分和顺式作用模块不能成为表达能力好的5'UTR，并在顺式作用模块的基础上删减元件得到了6条成功表达的5′UTR序列。值得一提的是从HSV-1即刻早期基因的5′UTR序列中发现验证了一条和β珠蛋白5'UTR能力相近的5'UTR以及一个未知的潜在元件，在目前通过人工设计得到表达能力强的5'UTR较为困难的情形下，在已知的序列里寻找表达能力强的5'UTR也是一种好的方法。 

mRNA vaccine has attracted much attention during the outbreak of novel coronavirus pneumonia due to its unique advantages. It uses human cells to produce viral antigen proteins that trigger an immune response. Compared with traditional vaccines, mRNA vaccines have many advantages, but their instability can greatly reduce the translation efficiency. The 5'UTR is an important structure that determines the expression efficiency of mRNA vaccines, and its secondary structure affects ribosome recruitment and initiation codon scanning. The researchers improved mRNA translation efficiency by optimizing the 5'UTR sequence. It is still a great challenge to design a 5'UTR with high protein expression, and it is also a good method to search for a 5'UTR with strong expression in known sequences. Since mRNA translation efficiency is complicated to be regulated by both 5'UTR and 3'UTR, and the object of this study was 5'UTR, all mrnas in this study did not have 3'UTR.

Herpes simplex virus type 1 (HSV-1), a member of the alpha-herpesvirus family, causes facial lesions in the mouth, lips, cornea and other parts of the eye, which cannot be completely cured and seriously affect the quality of life of patients. After HSV-1 invades host cells, the genes in the virus genome are regulated by the virus itself. One hour after infection, five immediate early genes, which have similar sequences upstream, were first rapidly expressed. By referring to a large number of literatures and comparing sequences, we found that there were cis-acting modules and a large number of elements in the first half of the immediate early gene upstream non-coding sequence, and the second half was the 5'UTR sequence obtained from the gene sequence of HSV-1 strain 17 (NC_001806.2) mRNA sequence obtained by Genbank. After knocking out immediate early genes RL2, RS1 and US1, it was found that the expression levels of early and late genes were abnormally increased at 1.5 hours after infection. The difference in the expression timing of viral proteins seemed to be determined by these special upstream non-coding sequences, which could reflect the importance of the non-coding sequences of HSV-1 immediate early genes.

Based on these studies, a question was raised as to how the two parts of the upstream non-coding sequence of the immediate early gene, respectively, as the 5'UTR of the mRNA of the model protein, affect its expression. In order to solve this problem, in the first part of this experiment, enhanced green fluorescent protein was used as the model protein to detect the effect of the first half of the upstream non-coding sequence of immediate early genes RL2 and RS1 as the 5'UTR of their mRNA on the expression level, and the results showed that the model protein was not expressed at all. Considering that the upstream non-coding sequences of RL2 and RS1 distribute cis-acting modules and each module is distributed with a large number of components, we took the three cis-acting modules of WTA, WTD and WTE as the 5'UTR of pattern protein mRNA, and found that the pattern protein was also not expressed. On this basis, 13 sequences were obtained according to the component distribution of the three cy-acting module sequences. 13 sequences were taken as the 5'UTR of pattern protein mRNA, among which 6 sequences WTA1, WTA2, ASP1, DSP1, Degr and KLF had different levels of pattern protein expression, which had certain practical value. In the first part of the experiment, it was concluded that the first half of the upstream non-coding sequence of the immediate early gene did not have the 5'UTR capability as an mRNA vaccine, but it was indisputable that the expression level of the immediate early gene increased rapidly at the beginning of infection. Considering that between the first half of the upstream non-coding sequence and the coding sequence is the 5'UTR of the immediate early gene itself, whether these 5'UTR mRNA 5'UTR with good expression level is worth exploring. In the second part of the experiment, the 5'UTR of RL2, RS1, US1 and US12 was used as the 5'UTR of model protein mRNA. mRNA transfection reagent and nano-liposome particle delivery methods were used to detect the differences in the expression levels of model protein in vitro cells and in animals, respectively. The expression level of RL2's 5'UTR was close to that of the control βglobin 5'UTR, and the expression level of RS1's 5'UTR was slightly weaker than that of the control 5'UTR. The 5'UTR of RL2 has the potential to become the 5'UTR of mRNA vaccine. We analyzed the 5'UTR expression of RS1, US1, US12, which was weaker than RL2. On the one hand, EGFP mRNA with RL2 5'UTR was more stable; On the other hand, we found repeated gcatcg sequences on the 5'UTR of RS1, US1 and US12 by sequence comparison.

The repeated occurrence of gcatcg sequences in immediate early gene non-coding sequences has aroused our interest, and the existence of this sequence may be related to the rapid expression of immediate early genes. In the third part of the experiment, the deletion of gcatcg near the starting codon position was sequentially observed to observe the effect of sequentially deletion of gcatcg on the expression of model protein. The results showed that deletion of the first gcatcg sequence resulted in a pattern protein level similar to the 5'UTR of βglobin, and deletion of two gcatcg sequences resulted in no pattern protein expression at all. Based on this result, we boldly speculated that gcatcg or sequences containing gcatcg may be an undiscovered potential element, and the presence of one gcatcg in the immediate early gene 5'UTR sequence is conducive to mRNA expression, while the presence of two gcatcg can reduce mRNA expression level, but this result still needs further verification.

The 5'UTR sequence is important for both mRNA therapy and vaccine development. Although a small number of Kozak-like elements have been found on the 5'UTR, there has not been much progress in improving foreign protein expression. In this study, it was preliminarily proved that the first half of the upstream non-coding sequence of RL2 and RS1 and the cis-acting module could not be a 5'UTR with good expression ability, and six successfully expressed 5'UTR sequences were obtained by deleting elements from the cis-acting module. It is worth mentioning that from the 5'UTR sequence of HSV-1 immediate early gene, a 5'UTR similar to βglobin's 5'UTR and an unknown potential element were found and verified. At present, it is difficult to obtain a 5'UTR with strong expression ability through artificial design. It is also a good idea to look for expressive 5'UTR in known sequences.