将含重组白细胞介素12（hIL-12）的杆状病毒（Ac-hIL12）经空斑纯化后，在草地贪夜蛾Sf9细胞中进行连续无稀释传代到P55代，收集被P15、P25、P35、P45、P55代重组病毒感染的细胞，抽提胞内病毒（ICV）DNA。根据重组病毒构建的原理，在P35 cDNA和P40 cDNA的3末段设计一对引物进行PCR，扩增出了包括P35 cDNA、Polyhedrin启动子、P10启动子和P40 cDNA序列在内的全长约2.0kb片段，克隆至T Vector进行序列测定后发现，在第P15、P25和P35代所扩增出的序列没有发生任何突变。但在P45代，P35cDNA中就有3个碱基发生了点突变（461T→C，517A→G以及630C→T），Polyhedrin启动子的+1位后插入了一个碱基T，P40 cDNA与P10启动子区（230bp）没有变化；而第P55代除了以上碱基突变以外，P10启动子区-168位的G替换突变为T， -136与-135位之间插入一个碱基T，以及-122位缺失一个碱基T。以上结果表明杆状病毒在体外细胞连续传代过程中可导致外源基因本身的突变。

The strain purified from a baculovirus expressing recombinant human interleukin-12 by plaque selection was serially and undilutedly passaged up to 55th generation in Spodoptera frugiperda 9(Sf9) cells. Intracellular viral (ICV) DNAs were extracted from the infected Sf9 cells of P15, P25, P35, P45, P55 generation of recombinant baculovirus. A 2.0kb DNA fragment including sequences of P35 cDNA, polyhedrin promoter, P10 promoter and P40 cDNA was amplified by PCR. The sequence analysis indicates that there is no mutation in 2.0kb nucleotide sequence during the P15 to P35 passages. However, the point mutation was detected at three nucleotide residue sites in P35 cDNA sequence (461T→C，517A→G and 630C→T) and one nucleotide “T” was inserted between +1 position of polyhedrin promoter and the upstream of BamH I recoganizing site at P45 generation. When passaging to P55, the insertional mutation (-136T-135), deleton mutation (-122T) and point mutation (-168G→T) happened in the P10 promoter cassette besides the above mutations. These results show that serial and undiluted passaging of engineering baculovirus can result in foreign gene mutation.