Cloning Of The Full-length CDNA Genome Of Sendai Virus BB1 Strain And Construction Of SeV Infectious Clone Expression Vector

Sendai virus is an enveloped nonsegment-negative strand RNA virus belonging to the order paramyxoviridae, paramyxoviridae family, respiroratory subfamily and hosting in human beings and mice. Its genome contains six ORFs coding for main polypeptides including nucleoprotein, phosphoprotein, matrix protein, fusion protein, hemagglutinin neuraminidase and the large protein with an untranscriptional region of 119bp at RNA 3' terminus called leader and an untranscriptional region of 139bp at 5' terminus called trailer. The genomic RNA structure of sendai virus is 3 ' -Leader-N-P-M-F-HN-L-Trailer-5' .In the year 1994, Schnell et al reported for the first time the successful rescue of rabies virus by reverse genetics, which opened a new era of negative strand RNA virus research. In 1997, Kato et al first succeeded in rescuing the sendai virus Z strain by the reverse genetics and then the Nagoyo strain and the Fushimi strain were also rescued. The sendai virus BB1 strain used in our study was first isolated from the lung of the infected mice by HOU Yun De in Moscow in the 1960's.In our previous work, Yang Yu et al finished sequencing the whole genome of the sendai virus BB1 strain for the first time and submitted it to GenBank with an access number DQ219803. It shows that the sendai virus BB1 strain could be classified as an independent genotype which is neither homogenious to that of the Ohita strain and Hamamatsu strain nor to that of the Nagoya strain, pi strain and the Z strain.We started our work from sequencing the four overlapping fragments named A,B,C and D, covering the whole genome of sendai virus BB1 strain. The L gene in pDC-316 were also sequenced. The sequencing results with T7 and M13R in pCR-XL-TOPO show that the sequences of A,B ,D and L are as expected, while a large deletion occurs in C fragment which covers nt7794-8270 in reality. So we decided to link the whole cDNA genome with A,B ,D and L. However, there still existed a gap from nucleotide 8294-8555.We synthesized the above fragment artificially by the coretemplate method. Unluckily, we found another deletion of G in this synthetical fragment at nt 8463. Then we corrected the G deletion by the method of reverse PCR and renamed the right fragment containing the deleted 8294-8555 sequence as C1. Inserting C1 fragment into pCR-XL-TOPO-B-1 we got BC1. After adding an enzymatic site SmaI at the 5' terminus of the A fragment we got A1, and then it was ligated into BC1 obtaining A1BC1 fragment which ranges from nt 1 to 8555 of the 5' terminus of BB1 sendai virus cDNA genome. Then we got the L-Trailer fragment abbreviated as LT which ranges from nt 8556-15384 by linking L with the Trailer in D fragment. The whole genome is now at the two-fragment stage: A1BC1 and LT. We chose the plasmid vector pBluescirpt II-KS(+) for linking the whole cDNA genome of the BB1 strain. We firstly cloned LT into pBluescirpt II KS(+) obtaining pBluescrit II-KS-LT(abbreviated as pBKS-LT), and then ligated the A1BC1fragmet into pBKS-LT and created the plasmid pBluescirpt II KS-SeV BB1 containing the whole cDNA genome of the sendai virus BB1 strain.We also studied the SeV infectious exression vector at the same time. Firstly, we sequenced the sendai virus minigenome vector named pminiSeV-EGFP/TM1 which was constructed by Yang Yu. The results showed that the actual key elements arrangement in pminiSeV-EGFP/TM1 is as follows: HdvRz-Leader-EGFP-Trailer-HamRz. We also found missing of two loops in the hepatitis delta virus ribozyme. Uncertain about the impact of the missing, we corrected the mutation by PCR to creat pminiSeV-EGFP/ TM1m and sequenced for confirmation. Then we designed and constructed the pVAX-SeVV-EGFP(-) plasmid with CMV as its promoter. Its structure is CMVpro- HamRz-Trailer-EGFP(-)- Leader-HdvRz-polyA, in which EGFP(-) represents the EGFP gene ORF's direction is reverse to transcriptional direction of CMV promoter. This means that the reporter gene will not be expressed directly from the transcription of CMV promoter. BHK-21 cells transfected with pVAX- SeVV- EGFP(-) observed no GFP expression. While those transfected cells with the help of sendai virus infection showed GFP expression as well as cell fusion. This indicates that the minigenome of SeV in pVAX-SeVV-EGFP(-) was rescued and translated efficiently. At the end, we designed and constructed an universal SeV infectious vector with arrangement of CMVpro- HamRz-Leader-MCS-Trailer-HdvRz-polyA. The full length genome between N and L will be inserted into pVAX-SeVV to generate infectious sendai virus by cotransfecting sensitive cells with the plasmids expressing N,P and L.